UCSF
UC San Francisco Previously Published Works
Title
Genetic and functional characterization of clonally derived adult human brown
adipocytes
Permalink
https://escholarship.org/uc/item/4qk4m480
Journal
Nature Medicine, 21(4)
ISSN
1078-8956
Authors
Shinoda, K
Luijten, IHN
Hasegawa, Y
et al.
Publication Date
2015-04-01
DOI
10.1038/nm.3819
Peer reviewed
eScholarship.org Powered by the California Digital Library
University of California
© 2015 Nature America, Inc. All rights reserved.
l e t t e r s
nature medicine ADVANCE ONLINE PUBLICATION 1
Brown adipose tissue (BAT) acts in mammals as a natural
defense system against hypothermia, and its activation to a
state of increased energy expenditure is believed to protect
against the development of obesity. Even though the existence
of BAT in adult humans has been widely appreciated
1–8
, its
cellular origin and molecular identity remain elusive largely
because of high cellular heterogeneity within various adipose
tissue depots. To understand the nature of adult human
brown adipocytes at single cell resolution, we isolated clonally
derived adipocytes from stromal vascular fractions of adult
human BAT from two individuals and globally analyzed their
molecular signatures. We used RNA sequencing followed by
unbiased genome-wide expression analyses and found that a
population of uncoupling protein 1 (UCP1)-positive human
adipocytes possessed molecular signatures resembling those
of a recruitable form of thermogenic adipocytes (that is, beige
adipocytes). In addition, we identified molecular markers that
were highly enriched in UCP1-positive human adipocytes,
a set that included potassium channel K3 (KCNK3) and
mitochondrial tumor suppressor 1 (MTUS1). Further, we
functionally characterized these two markers using a loss-of-
function approach and found that KCNK3 and MTUS1 were
required for beige adipocyte differentiation and thermogenic
function. The results of this study present new opportunities
for human BAT research, such as facilitating cell-based disease
modeling and unbiased screens for thermogenic regulators.
Recent studies using
18
fluoro-2-deoxyglucose positron emission tom-
ography (
18
FDG-PET) scanning demonstrated that the prevalence
of adult human BAT is inversely correlated with body mass index
(BMI), adiposity, and fasting plasma glucose level
1–6
, indicating that
BAT is likely to play a role in human metabolism. Current evidence
indicates that two types of UCP1-positive thermogenic adipocytes
exist in rodents and humans: classical brown adipocytes and beige
adipocytes (also known as brite adipocytes). Classical brown adi-
pocytes arise from a subset of the dermomyotome during embryonic
development
9–12
. They are found predominantly in adipose depots of
rodents and infants that are mostly dedicated to BAT, such as those in
the interscapular regions. Beige adipocytes, on the other hand, reside
mainly in subcutaneous white adipose tissues (WAT), where they arise
postnatally in response to certain external cues—such as chronic
cold exposure or long-term treatment with agonists of peroxisome
proliferator-activated receptor-γ (PPAR-γ)—a process often referred
to as the ‘browning’ of WAT
13–17
.
Although previous studies have reported that adult human BAT
possesses a molecular signature resembling that of mouse beige
adipocytes
16,18,19
, more recent data imply that cultured adipocytes
derived from adult human BAT express several classical brown
adipocyte–selective markers that were originally found in mice
20
. This
discrepancy appears to be primarily due to a few reasons. First, adult
human BAT is a highly heterogeneous tissue compared to mouse BAT,
consisting of UCP1-positive multilocular brown adipocytes, UCP1-
negative unilocular white adipocytes, endothelial cells, stromal cells,
and immune cells. Indeed, the gene-expression profile of human BAT
in the neck region varies depending on the depth of the tissue
21
. Hence,
molecular analyses of biopsied adult human BAT samples could be
confounded by potential contamination from UCP1-negative cells
such as white adipocytes and myocytes. Second, conclusions made in
previous studies were entirely based on the mRNA-expression profiles
of a few selected genes that were originally identified in mice. Global
and unbiased molecular analyses in a homogeneous cell population
are therefore warranted to clarify the nature of adult human BAT.
The discrepancy regarding the cellular identity of adult human BAT
needs to be critically assessed to make it possible to strategize future
therapeutic interventions for anti-obesity treatment through targeting
this tissue. Namely, the identification of human-specific BAT molec-
ular markers will allow for the development of cell type–selective
activators that are likely to act more effectively and more safely than
Genetic and functional characterization of clonally
derived adult human brown adipocytes
Kosaku Shinoda
1–3,12
, Ineke H N Luijten
1–4,12
, Yutaka Hasegawa
1–3
, Haemin Hong
1–3
, Si B Sonne
1–3,11
,
Miae Kim
1–3,11
, Ruidan Xue
5,6
, Maria Chondronikola
7–10
, Aaron M Cypess
5,6
, Yu-Hua Tseng
5,6
, Jan Nedergaard
4
,
Labros S Sidossis
7–10
& Shingo Kajimura
1–3
1
Diabetes Center, University of California, San Francisco (UCSF), San Francisco, California, USA.
2
Department of Cell and Tissue Biology, UCSF, San Francisco,
California, USA.
3
Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, UCSF, San Francisco, California, USA.
4
Department of Molecular
Biosciences, The Wenner-Gren Institute, Stockholm University, Stockholm, Sweden.
5
Joslin Diabetes Center, Boston, Massachusetts, USA.
6
Harvard Medical
School, Boston, Massachusetts, USA.
7
Metabolism Unit, Shriners Hospitals for Children, Galveston, Texas, USA.
8
Department of Internal Medicine, University
of Texas Medical Branch, Galveston, Texas, USA.
9
Department of Surgery, University of Texas Medical Branch, Galveston, Texas, USA.
10
Department of Nutrition
and Metabolism, University of Texas Medical Branch, Galveston, Texas, USA.
11
Present addresses: Department of Biology, University of Copenhagen, Copenhagen,
Denmark (S.B.S.), East Coast Life Sciences Institute, Gangneung-Wonju National University, Gangneung, South Korea (M.K.).
12
These authors contributed equally to
this work. Correspondence should be addressed to S.K. (skajimura@diabetes.ucsf.edu).
Received 16 October 2014; accepted 6 February 2015; published online 16 March 2015; doi:10.1038/nm.3819
© 2015 Nature America, Inc. All rights reserved.
l e t t e r s
2 ADVANCE ONLINE PUBLICATION nature medicine
non-specific activators in recruiting new thermogenic adipocytes,
especially in subjects who do not possess appreciable levels of exist-
ing BAT. For example, even though non-selective pharmacological
activation of the sympathetic nervous system via beta3-adrenoceptor
agonists can activate both classical brown adipocytes and beige
adipocytes, adverse effects on the cardiovascular system prevents
the clinical use of these agonists
22
.
To this end, we have isolated a total of 65 clonally immortalized
preadipocyte lines from stromal vascular fractions (SVFs) obtained
from UCP1-positive supraclavicular BAT biopsies of two non-obese
individuals
20
. We found seven clonal lines (10.8%) among the clones
isolated from these biopsies that exhibited highly adipogenic prop-
erties. This result was similar to that of a previous report showing
that approximately 6.5% of cloned SVFs (20 out of 305 clonal lines)
displayed adipogenic properties in culture
16
. We subsequently ana-
lyzed three out of the seven adipogenic lines for RNA sequencing
(RNA-seq) and bioinformatics analyses. We also examined UCP1
mRNA expression of the seven adipogenic clones in response to
cAMP and to rosiglitazone (Supplementary Fig. 1a,b).
In order to study and identify the gene signatures of clonal
brown adipocytes, we experimentally differentiated the three clonal
preadipocyte cell lines into brown adipocytes under an adipogenic
culture condition. We took this approach because it is not possible to
immortalize clonally isolated brown adipocytes directly from human
biopsy material because adipocytes are post-mitotic cells. These experi-
mentally derived brown adipocytes cannot be formally construed as
a cell line (that is, they cannot be serially passaged while maintaining
their differentiated state), so we refer to them here as brown adipocyte
cultures 1–3. Additionally, because matched WAT pairs from the same
subjects were not available, we isolated 35 clonal preadipocyte lines
from SVFs of subcutaneous WAT from a lean BMI-matched individual
as a control, using the same immortalization protocol as for the
brown preadipocyte lines to avoid potential contamination from
SV40 large T antigen–mediated immortalization. The clonally derived
preadipocyte lines from subcutaneous WAT were differentiated under
the same cultured conditions as the clonal brown adipocyte cultures.
We refer to them as white adipocyte cultures 1–3.
We found that these three clonal preadipocyte cell lines, upon dif-
ferentiation, were highly adipogenic, as assessed by Oil-Red-O staining
(Fig. 1a). We further found that after experimentally induced differen-
tiation, brown adipocyte cultures 1–3 showed significantly (P < 0.001)
higher expression of UCP1 and PPARGC1A (encoding PPAR-γ coac-
tivator 1α) mRNA, both in the basal state and after a 4 h–long treat-
ment with cAMP before harvesting, as compared to white adipocyte
cultures 1–3 (Fig. 1b). The UCP1 mRNA expression in the three brown
adipocyte cultures after cAMP treatment was similar to that found in
biopsied BAT samples isolated from the supraclavicular region, whereas
such induction was not seen in the white adipocyte cultures (Fig. 1b).
The induction of PPARGC1A expression upon cAMP stimulation in the
brown adipocyte cultures, however, did not reach the levels seen in the
biopsied BAT control, although it was still potent (Fig. 1b).
UCP1 protein expression was also detected in the three brown
adipocyte cultures, but not in the white adipocyte cultures. We
observed higher UCP1 expression in brown adipocyte cultures treated
with cAMP and rosiglitazonethan in the vehicle-treated cells (Fig. 1c).
Notably, the differentiated clonal brown adipocyte cultures showed
greater total and uncoupled cellular respiration in both the basal
and the cAMP-stimulated states than in the clonal white adipocyte
cultures with similar adipogenic properties (Fig. 1d). Induction of
greater total and uncoupled cellular respiration was not observed
in the brown preadipocyte cell lines (Supplementary Fig. 1c).
Furthermore, the differentiated clonal brown adipocyte cultures
Figure 1 Isolation of clonal brown adipocytes
from adult human BAT. (a) Representative
Oil-Red-O staining of differentiated brown
adipocyte cultures 1–3 and white adipocyte
culture 1 at low magnification (top) and at
high magnification (bottom). n = 3 for all
groups. Scale bars, 50 µm. (b) Expression of
UCP1 (top) and PPARGC1A (bottom) in the
differentiated clonal brown adipocyte cultures
1–3 and white adipocyte cultures 1–3 treated
with forskolin (cAMP) or vehicle (basal). BAT,
biopsied human BAT from the supraclavicular
regions (positive control). mRNA expression
relative to expression of housekeeping gene
TBP. n = 3 for all groups.
§§§
P < 0.001, brown
versus white adipocyte lines; *P < 0.05,
**P < 0.01, ***P < 0.001, brown or white
(as indicated) versus basal by one-sided
Welch’s t-test. The error bars for UCP1 in
white adipocytes are 0.001, 0.002 and
0.001 in cultures 1, 2 and 3, respectively.
(c) Western blot of UCP1 in differentiated
brown adipocyte culture 1 and white adipocyte
culture 1 treated with forskolin (cAMP) or
rosiglitazone. β-actin, loading control.
Data are representative of two experiments.
(d) Total and uncoupled cellular respiration
in differentiated brown adipocyte culture 2
and white adipocyte culture 1 treated with
forskolin (cAMP) or vehicle (basal).
OCR, oxygen consumption rate. n = 8 for all groups. ***P < 0.001 by one-sided Student’s t-test. NS, not significant. The variance was similar
between basal and cAMP groups (P = 0.709). Data are expressed as means ± s.e.m. for all bar graphs.
Basal
***
***
**
§§§
3
3
4
2
2
Brown White
BAT
1
1 321
0
Relative UCP1 expression
cAMP
***
**
§§§
**
**
*
Brown White
BAT
60
15
10
5
0
1 2 3 1 2 3
Relative PPARGC1A expression
Total
Uncoupled
100
80
60
40
20
0
***
Mitochondria-derived OCR
(pmol min
–1
)
Brown 2 White 1Brown 2 White 1
Basal
cAMP
NS
NS
***
cAMP
–
+
+
+
+– –
– –
– –
–
Rosiglitazone
UCP1
β-actin
Brown 1
a
c
d
b
Brown 2 Brown 3 White 1
Brown 1 White 1
© 2015 Nature America, Inc. All rights reserved.
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nature medicine ADVANCE ONLINE PUBLICATION 3
possessed cAMP-induced lipolysis capacity, as glycerol release was
higher in the cells treated with forskolin, a selective activator of the
enzyme adenylyl cyclase, as well as in the cells treated with a cell-
penetrant cAMP analog, dibutyryl-cAMP than in the vehicle-treated
cells (Supplementary Fig. 1d). These data indicate that the three
clonal human adipocyte cultures exhibited molecular and functional
characteristics of thermogenic adipocytes.
To identify gene signatures that were distinct between human brown
and white adipocytes, we employed RNA-seq of clonal brown adi-
pocyte cultures 1–3 and clonal white adipocyte cultures 1–3 (Fig. 2a).
A complete list of the genes is provided in Supplementary Table 1.
To determine if differentiated human brown adipocytes were similar
or different to mouse classical brown adipocytes or beige adipocytes,
we overlapped the top 800 differentiated human brown adipocyte–
enriched genes with mouse orthologs that were enriched in differenti-
ated classical brown and/or beige adipocytes as previously reported
16
(Fig. 2a). Microarray data sets from clonally derived immortalized
mouse beige and classical brown adipocyte cultures under a differen-
tiated state were obtained from the Gene Expression Omnibus (GEO;
accession no. GSE39562)
16
. In addition to our own human brown
and white adipocyte RNA-seq data sets, we included publicly avail-
able microarray data sets of primary differentiated human adipocytes
(referred to here as primaries A, B, and C) derived from subcutaneous
WAT (GEO accession no. GSE41223; Fig. 2b). Hierarchical clustering
revealed that the three differentiated clonal human brown adipocyte
cultures belonged to a cluster of mouse beige adipocytes but were
distinct from a cluster of mouse classical brown adipocytes (Fig. 2b).
As an independent approach, principal component (PC) analysis
suggested that the gene-expression profiles of the three differenti-
ated clonal human brown adipocyte cultures were much closer to
those of mouse beige adipocytes than to those of mouse classical
brown adipocytes (relative Euclidean distance from human brown
adipocytes to mouse beige and classical brown adipocytes was 9.4. and
18.1, respectively) (Fig. 2c). These results suggest that the molecular
signatures of adult human brown adipocytes resemble those of mouse
beige adipocytes rather than mouse classical brown adipocytes.
Of note, RNA-seq analysis of the three clonal human preadipocyte
cell lines before differentiation revealed that the molecular signatures
of brown and white preadipocytes were distinct and formed separate
clusters (Fig. 2d). Consistent with a recent report
23
, we found that
the expression of contactin-associated protein-like 3 (CNTNAP3),
leucine-rich repeat-containing 17 (LRRC17), regulator of G-protein
signaling 7 binding protein (RGS7BP), phosphodiesterase 5A, cGMP-
specific (PDE5A), and ATP-binding cassette, subfamily A, member 9
(ABCA9) was higher in the clonal brown preadipocyte lines than
that in the clonal white preadipocyte lines (Supplementary Table 2).
0 0.6
Distance
Human primary A
Human primary B
Human primary C
Human white 3
Mouse beige A
Mouse beige B
Human brown 1
Human brown 3
Human brown 2
Mouse beige C
Mouse brown A
Mouse brown B
Mouse brown C
a c
d
–log
10
P value
7
6
5
Differentiated adipocytes
Preadipocytes
PPARGC1A
UCP1
DIO2
5%
4
3
2
1
0
–20 –15 –5
log
2
ratio (brown/white)
0 5 10 15 20–10
e
b
Brown
White
z-score
–0.7 0.7
Brown
White
z-score
–0.7 0.7
10
5
–5
–10
–10 –5 0 5 10
Principal component 1 (44.2%)
0
Principal component 2 (26.0%)
Mouse
Human
Brown
Beige
Brown
White
Figure 2 Genome-wide gene expression analyses
indicate a close relationship between human
brown adipocytes and mouse beige adipocytes.
(a) Expression profile and hierarchical clustering
of the differentially expressed genes between
differentiated clonal human brown adipocyte
cultures 1–3 and differentiated clonal white
adipocyte cultures 1–3 by two-fold or more.
n = 3 for each cell type. The color scale shows
z-scored FPKM (fragments per kilobase of exon
per million fragments mapped) representing
the mRNA level of each gene in a blue (low
expression)-white-red (high expression) scheme.
(b) Hierarchical clustering of human and mouse
adipocytes as visualized by TreeGraph. The
horizontal distance represents similarities among
each cluster. (c) Principal component (PC)
analysis of the transcriptome from human and
mouse differentiated adipocytes. PC analysis was
done using the same gene expression data set
used in b, that is, the RNA-seq data set obtained
from differentiated clonal human brown and
white adipocyte cultures, and the microarray
data set (GSE39562) from differentiated clonal
mouse classical brown and beige adipocytes.
Numbers in parentheses represent the proportion
of data variance explained by each PC.
(d) Expression profiles and hierarchical clustering
of the differentially expressed genes between
undifferentiated clonal human brown preadipocyte
cell lines 1–3 and white preadipocyte cell lines
1–3 by two-fold or more. n = 3 for each cell type.
The color scale is the same as in a. (e) Volcano
plot of transcriptomes in the clonal differentiated
human brown and white adipocyte cultures (red)
and in the clonal undifferentiated human brown
and white preadipocyte lines (blue). n = 3 for
each cell type. The log-fold change between
brown versus white is shown on the x-axis.
The y-axis represents the −log
10
of the P values
by delta method–based test. Previously defined
BAT-enriched markers are shown.
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4 ADVANCE ONLINE PUBLICATION nature medicine
This separation at the gene-expression level was further validated by
volcano plot analysis, which showed that the degree of difference in
brown and white adipocyte transcriptomes was comparable between
undifferentiated and differentiated states (Fig. 2e). These results indi-
cate that the cellular fate of brown versus white adipocytes is largely
determined at the precursor cell stage in adult humans.
From a developmental standpoint, it has been reported that mouse
beige adipocytes, but not classical brown adipocytes, express smooth
muscle lineage–selective genes and that a subset of beige adipocytes
arises from smooth muscle myosin, heavy chain 11, (Myh11)-positive
smooth muscle–like precursors
24
. Indeed, the isolated clonal human
brown preadipocyte lines abundantly expressed smooth muscle
lineage–selective genes, including ACTA2, TAGLN, MYL9, and CNN1, the
master smooth muscle transcription factor SRF, and the non-cardiac
co-activator MKL1 (Supplementary Fig. 2 and Supplementary
Table 3). These results further support the possibility that adult
human brown adipocytes exhibit beige-like characteristics. The
subcutaneous WAT–derived white preadipocyte lines also expressed
some of the smooth muscle lineage–selective genes, implying that
these cells may also possess ‘browning’ plasticity given appropriate
external stimuli.
To identify human brown adipocyte–selective markers, we ana-
lyzed the overlapped gene sets between human and mouse brown
adipocyte–enriched genes, which could be classified into four groups.
Group A contained genes that were enriched in the differentiated
clonal human brown adipocyte cultures as well as in mouse classi-
cal brown and beige adipocytes (Fig. 3a). Here we found previously
defined BAT markers, such as UCP1, PPARGC1A, EBF2 (ref. 25), and
BMPR1A
26
(Fig. 3b and Supplementary Table 4). The other groups
consisted of genes that were enriched in both human brown adipocytes
and mouse beige adipocytes (group B), genes enriched in both human
brown adipocytes and mouse classical brown adipocytes (group C),
and genes enriched only in human brown adipocytes (group D). The
subsequent gene ontology (GO) analysis found that the group A gene
set was enriched with genes encoding members of the metabolic path-
ways involved in BAT thermogenesis, such as the electron transport
chain, mitochondria biogenesis, cellular respiration, fatty acid oxi-
dation, lipid modification, and acetyl-CoA metabolism (Fig. 3c).
Next we validated the expression profiles of 36 representative human
brown adipocyte–enriched genes from biopsied human BAT sam-
ples
18
and integrated the correlation coefficient among these genes.
The correlation analysis found that a large number (31 out of 36;
86.1%) of these genes showed significant (P < 0.05) positive correla-
tions in expression (Supplementary Fig. 3a). We further validated
the expression of human BAT marker-gene candidates in the dif-
ferentiated clonal brown and white adipocyte cultures as well as in
3
2
1
0
3
2.5
2.0
1.5
1.0
0.5
0
2.5
2.0
1.5
1.0
0.5
0
2
1
0
3
2
1
0
3
10,000
6,000
3,000
30
25
20
15
10
5
0
2,000
1,000
200
100
0
2,000
150
100
WAT BAT WAT BAT
0
2
1
0
0 1 2 3 4
0 1 2 3 4
0 1 2 3 4
r = 0.679
r = 0.681
r = 0.513 r = 0.812
r = 0.772
r = 0.856
MTUS1
*
*
BAT
WAT
KCNK3
Relative PPARGC1A
expression
Relative
PRDM16
expression
Relative
MTUS1 expression
Relative KCNK3 expression
Relative CIDEA
expression
*
*
d e
GO analysis (Group A)
6
16
Electron transport chain
Mitochondrial biogenesis
Cellular respiration
Fatty acid oxidation
Lipid modification
Acetyl-CoA metabolism
Transmembrane transport
Coenzyme metabolism
11
4
5
5
6
9
7
Human
brown-enriched
genes
Group B (92)
a
c
b
Mouse
beige-enriched genes
Mouse
brown-enriched genes
Group C (65)
Group D
(1,499)
Group A (99)
UCP1
Brown
Human Mouse Human Mouse
White BrownWhite
White
Beige
Brown
White
Beige
Brown
PPARGC1A
BMPR1A
TNFRSF21
NDRG2
CYP26B1
EYA2
TRIM67
HSPH1
KTN1
HSPB7
ID3
TGOLN2
CYP4B1
SPTLC3
PDK4
KCNK3
REEP6
EBF2
PPARGC1B
LIFR
RNF34
FAM63B
MTUS1
EGLN3
TINAGL1
STAC2
SPARCL1
C10orf10
TNS2
DIO2
ANKRD28
SYNE2
C1orf52
HOXA2
MEOX2
CP
SFRP1
A
B
D
z-score
–0.7 0.7
C
PARM1
ANGPT4
PDGFRL
ORM1
CNR1
NDN
SLC7A2
FZD8
CLSTN2
TLR4
PTGIS
CD74
400
25
10
4
10
2
10
2
10
3
10
5
10
6
1
10
4
10
2
1
1
1 10
20
10
5
1
300
30
2
1
30 °C 19 °C 30 °C 19 °C
r = 0.64
r = 0.80
* *
MTUS1 expression
KCNK3 expression
Relative
MTUS1
expression
Relative
KCNK3
expression
Relative
UCP1
expression level
f
g
Relative MTUS1
expression
Relative KCNK3
expression
0 0.5 1.0 1.5 2.0 2.5
0 0.5 1.0 1.5 2.0 2.5
0 0.5 1.0 1.5 2.0 2.5
Figure 3 Identification of human brown
adipocyte markers. (a) Venn diagram of
the overlapping genes enriched in human
brown adipocytes, mouse classical brown
adipocytes, and mouse beige adipocytes
versus white adipocytes of the respective
species by two-fold or more. All cells were
differentiated clonal adipocytes in culture.
P < 0.05 by delta method–based test.
(b) Expression profiles of select genes enriched
in each group. The color scale shows z-scored
FPKM representing the mRNA level of each
gene in blue (low expression)-white-red (high
expression) scheme. (c) GO analysis of the
gene set in Group A (GO FAT category). The
area of each pie slice represents the number of
genes that belong to the indicated GO terms.
(d) Correlation between MTUS1 and KCNK3
mRNA expression on the x-axis and mRNA
expression of previously defined marker genes
PPARGC1A, PRDM16 and CIDEA on the y-axis.
mRNA expression relative to the housekeeping
gene TBP. n = 23 for each panel. P < 0.01
by z-test. (e) Gene expression of MTUS1 and
KCNK3 in UCP1-positive adipose tissues (BAT)
and UCP1-negative adipose tissues (WAT) from
the neck region of the same individuals (eight
pairs). *P = 0.017 and 0.044, respectively,
by Wilcoxon signed-ranked test. The right
bar graph shows the expression data without
normalization to each individual. Data are
expressed as means ± s.e.m. n = 8 for each
group, *P < 0.05 by one-sided Welch’s t-test.
(f) mRNA expression of MTUS1 and KCNK3
in the supraclavicular BAT isolated from
six subjects under thermoneutral conditions
(30 °C) and prolonged cold exposure (19 °C).
Expression relative to TBP. *P = 0.035 and
0.023, respectively, by Wilcoxon signed-ranked
test. (g) Correlation analysis between MTUS1
variant 3 or KCNK3 and UCP1 under prolonged
cold exposure. n = 13. P < 0.01 by z-test.