Thioesterase Superfamily Member 1 Suppresses Cold Thermogenesis by Limiting the Oxidation of Lipid Droplet-Derived Fatty Acids in Brown Adipose Tissue

Abstract

Objective:
Non-shivering thermogenesis in brown adipose tissue (BAT) plays a central role in energy homeostasis. Thioesterase superfamily member 1 (Them1), a BAT-enriched long chain fatty acyl-CoA thioesterase, is upregulated by cold and downregulated by warm ambient temperatures. Them1 (-/-) mice exhibit increased energy expenditure and resistance to diet-induced obesity and diabetes, but the mechanistic contribution of Them1 to the regulation of cold thermogenesis remains unknown.

Methods:
Them1 (-/-) and Them1 (+/+) mice were subjected to continuous metabolic monitoring to quantify the effects of ambient temperatures ranging from thermoneutrality (30 °C) to cold (4 °C) on energy expenditure, core body temperature, physical activity and food intake. The effects of Them1 expression on O2 consumption rates, thermogenic gene expression and lipolytic protein activation were determined ex vivo in BAT and in primary brown adipocytes.

Results:
Them1 suppressed thermogenesis in mice even in the setting of ongoing cold exposure. Without affecting thermogenic gene transcription, Them1 reduced O2 consumption rates in both isolated BAT and primary brown adipocytes. This was attributable to decreased mitochondrial oxidation of endogenous but not exogenous fatty acids.

Conclusions:
These results show that Them1 may act as a break on uncontrolled heat production and limit the extent of energy expenditure. Pharmacologic inhibition of Them1 could provide a targeted strategy for the management of metabolic disorders via activation of brown fat.

Full text

Thioesterase superfamily member 1 suppresses
cold thermogenesis by limiting the oxidation of
lipid droplet-derived fatty acids in brown
adipose tissue
Kosuke Okada
1
, Katherine B. LeClair
1
, Yongzhao Zhang
1
,
4
, Yingxia Li
1
, Cafer Ozdemir
1
, Tibor I. Krisko
1
,
Susan J. Hagen
2
, Rebecca A. Betensky
3
, Alexander S. Banks
1
, David E. Cohen
1
,
*
ABSTRACT
Objective: Non-shivering thermogenesis in brown adipose tissue (BAT) plays a central role in energy homeostasis. Thioesterase superfamily
member 1 (Them1), a BAT-enriched long chain fatty acyl-CoA thioesterase, is upregulated by cold and downregulated by warm ambient
temperatures. Them1
/
mice exhibit increased energy expenditure and resistance to diet-induced obesity and diabetes, but the mechanistic
contribution of Them1 to the regulation of cold thermogenesis remains unknown.
Methods: Them1
/
and Them1
þ/þ
mice were subjected to continuous metabolic monitoring to quantify the effects of ambient temperatures
ranging from thermoneutrality (30

C) to cold (4

C) on energy expenditure, core body temperature, physical activity and food intake. The effects
of Them1 expression on O
2
consumption rates, thermogenic gene expression and lipolytic protein activation were determined ex vivo in BAT and
in primary brown adipocytes.
Results: Them1 suppressed thermogenesis in mice even in the setting of ongoing cold exposure. Without affecting thermogenic gene tran-
scription, Them1 reduced O
2
consumption rates in both isolated BAT and primary brown adipocytes. This was attributable to decreased
mitochondrial oxidation of endogenous but not exogenous fatty acids.
Conclusions: These results show that Them1 may act as a break on uncontrolled heat production and limit the extent of energy expenditure.
Pharmacologic inhibition of Them1 could provide a targeted strategy for the management of metabolic disorders via activation of brown fat.
Ó2016 The Authors. Published by Elsevier GmbH. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Keywords Energy expenditure; Fatty acyl-CoA; Acyl-CoA thioesterase; Mitochondria; Obesity
1. INTRODUCTION
In response to cold exposure, activation of mitochondria-rich brown
adipose tissue (BAT) by norepinephrine (NE) signaling promotes non-
shivering thermogenesis [1]. Heat is generated when mitochondrial
b
-oxidation is uncoupled from oxidative phosphorylation by uncoupling
protein 1 (Ucp1). Key events include the liberation of intracellular tri-
glycerides from lipid droplets by activation of lipolysis. The released
free fatty acids (FFA) are channeled to mitochondria following their
conversion to fatty acyl-CoAs by long chain acyl-CoA synthetase (Acsl)
1[2].
Cold exposure also leads to transcriptional upregulation of a thermo-
genic gene profile in BAT [3,4]. Thioesterase superfamily member
(Them) 1 (synonyms: brown fat inducible thioesterase (BFIT), ste-
roidogenic acute regulatory protein-related lipid transfer (START)
domain 14 (StarD14)/acyl-CoA thioesterase 11 (Acot11)) was identified
as a BAT-enriched gene that was upregulated when mice were
exposed to cold and was suppressed by acclimation to warm ambient
temperatures [5]. Them1 is a type 2 acyl-CoA thioesterase [6,7], and
the recombinant protein catalyzes the hydrolysis of long chain fatty
acyl-CoA molecules into FFA plus CoASH [8,9]. Based on its tran-
scriptional upregulation in concert with thermogenesis and higher
1
Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA
2
Department of Surgery, Beth Israel Deaconess Medical Center,
Harvard Medical School, Boston, MA, USA
3
Department of Biostatistics, Harvard T.H. Chan School of Public Health, Boston, MA, USA
4
Present address: Envirologix, Inc., 500 Riverside Industrial Pkwy, Portland, ME 04103, USA.
*Corresponding author. Brigham and Women’s Hospital, 77 Avenue Louis Pasteur, HIM 941, Boston, MA 02115, USA. Tel.: þ1 (617) 525 5090; fax: þ1 (617) 525 5100.
E-mail: dcohen@partners.org (D.E. Cohen).
Abbreviations: Acot, acyl-CoA thioesterase; Ascl, long chain acyl-CoA synthetase; Atgl, adipose triglyceride lipase; AUC, area under the curve; BAT, brown adipose tissue;
ASM, acid soluble metabolites; BFIT, brown fat inducible thioesterase; CPT, carnitine palmitoyl transferase; Fabp, fatty acid binding protein; FCCP, carbonyl cyanide-p-
trifluoromethoxyphenylhydrazone; FFA, free fatty acids; Hsl, hormone sensitive lipase; PKC, protein kinase C; Plin, perilipin; MOI, multiplicity of infection; NE, norepinephrine;
OCR, oxygen consumption rate; Ppar, peroxisome proliferator-activated receptor; RER, respiratory exchange rate; START, steroidogenic acute regulatory protein-related lipid
transfer; Them1, thioesterase superfamily member; UCP, uncoupling protein; WAT, white adipose tissue
Received February 1, 2016

Revision received February 9, 2016

Accepted February 12, 2016

Available online 23 February 2016
http://dx.doi.org/10.1016/j.molmet.2016.02.002
Original article
340 MOLECULAR METABOLISM 5 (2016) 340e351 Ó2016 The Authors.Published by Elsevier GmbH.This is an open access article under theCC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
www.molecularmetabolism.com

expression in BAT of obesity resistant mouse strains, it has been
postulated that Them1 promotes energy expenditure [5,10]. However,
Them1
/
mice exhibit increased energy expenditure at room tem-
perature, along with resistance to high fat diet-induced obesity and
glucose intolerance, suggesting that Them1 may instead limit the
thermogenic capacity of BAT [11]. Although the mechanism is un-
known, reduced acyl-CoA thioesterase activities of BAT homogenates
from Them1
/
mice taken together with increased tissue concen-
trations of long chain fatty acyl-CoAs suggest that Them1 functions
endogenously as a fatty acyl-CoA thioesterase [9,11]. This study
examined Them1-mediated regulation of energy expenditure when
mice were challenged with cold stress and the mechanism responsible
for this effect. The results demonstrate that Them1 in BAT suppresses
non-shivering thermogenesis by limiting the oxidation of lipid droplet-
derived fatty acids. We propose that Them1 in BAT directly regulates
the availability of substrates to be utilized for
b
-oxidation and
uncoupling.
2. METHODS
2.1. Animals and diets
Male 5 to 13 w-old Them1
/
and littermate Them1
þ/þ
mice [11]
were housed in a barrier facility with free access to water and a
standard chow diet (PicoLab Rodent Diet 20, 5053, LabDiets; St. Louis,
MO, USA). The ambient temperature was maintained at 22 1

C and
the light and dark cycles were each 12 h (light cycle: 6 AMe6 PM, dark
cycle: 6 PMe6 AM). Body composition was determined by NMR
spectroscopy using an EchoMRI 3-in-1 Body Composition Analyzer
(Houston, TX, USA). For experiments designed to measure core body
temperature, mice were surgically implanted with small intraperitoneal
temperature transponders (Mini Mitter, Bend, OR, USA) as previously
described [12], and allowed a 1 w recovery period prior to experiments.
In order to harvest tissue and plasma, mice were fasted for 4 h and then
euthanized using CO
2
. Samples were snap-frozen in liquid nitrogen and
stored at 80

C. All protocols for animal use and euthanasia were
approved by the institutional committee of Harvard Medical School.
2.2. Comprehensive mouse monitoring
Experiments were performed using a temperature- and light (12 h
light/dark cycle)-controlled Comprehensive Lab Animal Monitoring
System (CLAMS, Columbus Instruments; Columbus, OH, USA) essen-
tially as described [12]. Briefly, 9e12 w-old mice were placed in in-
dividual cages without bedding, but with free access to chow and
water. Rates (ml/kg/h) of O
2
consumption (VO
2
) and CO
2
production
(VCO
2
), as well as core body temperatures were determined at 11 min
intervals. Respiratory exchange ratio (RER) values were calculated as
VCO
2
/VO
2
. Rates of energy expenditure (kJ/h) were calculated from gas
exchange. Activity was determined according to beam breaks and food
intake was measured gravimetrically.
Each mouse was weighed prior to being placed in the CLAMS. Mice were
studied at ambient temperatures ranging from 30

Cto4

C according
to two different experimental designs [12]. Under “temperature-
dynamic conditions”, mice were first acclimated to 30

C for 24 h in
the CLAMS prior to 24 h of data recording. The ambient temperature was
then reduced sequentially every 24 h. Data were recorded continuously,
without further acclimation periods. During the course of these exper-
iments, mice were not removed from their cages to determine
temperature-dependent body weights and compositions, which were
instead calculated based on the initial weights of the mice and average
temperature-dependent rates of weight loss and body compositions
determined in separate experiments. For “temperature-equilibrated”
conditions, mice were housed for 96 h at each ambient temperature,
with 48 h for acclimation, and then 48 h for data collection. Body weights
and composition were determined at the beginning and end of exposure
to each ambient temperature and averaged to yield values for each
recording period. Because mice implanted with metal-containing
temperature transponders could not be subjected to NMR de-
terminations of body composition, separate cohorts of mice were uti-
lized for determinations of core body temperature.
For each ambient temperature, cumulative values of energy expendi-
ture were adjusted for differences in lean body weight and compared
by analysis of covariance (ANCOVA) [12,13] using VassarStats (www.
vassarstats.net). We fit separate linear mixed effects regression
models for rates of energy expenditure at each ambient temperature
for the temperature-dynamic experiments and the temperature-
equilibrated experiments. Random effects were included for mice to
adjust for correlation within groups, and fixed effects for genotype, lean
body weight, dark versus light and the interaction of genotype with
dark versus light. Least squares means estimates of rates of energy
expenditure were calculated for each genotype and dark/light com-
bination to adjust for lean body weight. Similar mixed effects models
were fit for core body temperature, total activity and ambulatory ac-
tivity, without adjustment for lean body weight. SAS Version 9.3 (SAS
Institute Inc., Cary, NC, USA) was utilized to implement these analyses.
In a separate experiment, mice were acclimated to 30

C overnight in
the CLAMS. VO
2
values were then recorded for 120 min, after which
mice were injected (0.5 mg/kg) with the selective
b
3
-adrenergic re-
ceptor agonist CL316,243 (Sigma Chemical Co. St Louis, MO, USA)
[14]. Mice were returned immediately to the CLAMS and values of VO
2
recorded for an additional 280 min. To measure the response of BAT
gene expression to CL316,243, similarly treated mice were sacrificed
60 or 90 min following injection. For gene expression studies, mice
injected with an equal volume of PBS and sacrificed after 90 min
served as controls.
2.3. Surface infrared thermography
Thermal radiation was assessed in mice housed for 48 h at ambient
temperatures ranging from 30

Cto4

C using an FLIR Systems model
T420 infrared camera (Wilsonville, OR, USA) as described [15], with
minor modifications. To eliminate stray light, the FLIR camera was
mounted on a box and placed over a custom-designed metal grid at
focal length of 30 cm. Immediately upon removal from their
temperature-controlled environment, mice were anesthetized with
isoflurane and then placed in the prone position on the metal grid.
Separate images were taken of the back and the tail. To enable
specific imaging of the proximal tail, handling of mice was limited to
the distal-most tip of the tail, which was placed on a dark felt support.
Thermal images were captured at emissivity setting of 0.95 in JPG
format. Images were converted to semicolon-delimited. CSV files using
the FLIR ResearchIR program. Files were then batch converted to RAW
format using Matlab (The Mathworks, Natick, MA, USA) (script kindly
provided by J. Crane & G. Steinberg, Macmaster Univ., Ottowa, Ontario,
Canada), which were imported into AMIDE image analysis software
(http://www.amide.sourceforge.net) with the following specified
format: “float, little endian 32-bit,”0 offset bytes, 240 320 1
dimensions (specific to the camera model) with gate, frames and voxel
size each set to 1. For interscapular or tail imaging, digital fiducial
markers were placed at the midline and rearward base of the ears or
base of tail, respectively, and referenced to align and overlap each
image. For the interscapular area, the region of interest (ROI) was
placed as 24.5 mm aligned with the long axis of mouse 28.0 mm
beginning at the rearward base of the ears and centered on the
MOLECULAR METABOLISM 5 (2016) 340e351 Ó2016 The Authors.Published by Elsevier GmbH. This is an open access articleunder the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
www.molecularmetabolism.com 341

scapulae. For the tail, a ROI 24.0 mm 50.8 mm was placed over the
tail, beginning at its base and centered on the long axis of the tail.
Absolute scaling of the ROI was confirmed using the custom-designed
grid spacing. For each ROI, the mean of the warmest 10% of voxels
was calculated using Amide and designated to be the thermal radiation
value for the sample.
2.4. Biochemical analyses
Concentrations of FFA and triglycerides in tissue, cells and plasma
were determined using reagent kits (Wako Chemicals; Richmond, VA,
USA). Plasma glycerol concentrations were determined using a free
glycerol determination kit (SigmaeAldrich; St. Louis, MO). Plasma
glucose concentrations were determined using a OneTouch Ultra
glucose monitor (LifeScan, Inc.; Milpitas, CA, USA).
2.5. Histology and ultrastructure
Freshly harvested BAT from 12 to 13 w-old mice was immersed in
Bouin’sfixative solution. Hematoxylin and eosin staining was per-
formed by the Histology Core at the Beth Israel Deaconess Medical
Center, Harvard Medical School, Boston, MA. Electron microscopy was
performed as previously described [12]. Following fixation, dehydra-
tion, and embedding in LX112 resin (Ladd Research Industries; Bur-
lington, VT), ultrathin BAT sections were prepared using a Leica
Ultracut E ultramicrotome (Leica Microsystems; Deerfield, IL). These
were visualized with a JEOL 1400 electron microscope (JEOL USA:
Peabody, MA, USA), and images taken using with a GatanCCD camera
(Gatan; Warrendale, PA, USA).
2.6. Culture and differentiation of primary brown adipocytes
Primary brown preadipocytes were isolated, cultured, and then differ-
entiated as described [12]. Briefly, BAT harvested from 5 to 7 w-old mice
(n ¼7/group) was pooled, minced, digested with collagenase (Sigmae
Aldrich) and dispersed in DMEM containing 4.5 g/L glucose, 0.1 mM
pyruvate, 10 mM HEPES, 1% penicillin-streptomycin and 1
m
g/ml
fungizone. Unless otherwise specified, media contained 10% FBS. After
5e6 d, cells were trypsinized and seeded at 25,000 cells/cm
2
. When
cells achieved confluence, differentiation of the preadipocytes was
initiated by addition to the media of 1
m
M rosiglitazone (SigmaeAldrich)
for 7 d [16]. This method was chosen because preliminary experiments
revealed greater Them1 and Ucp1 expression than observed using the
combination of isobutylmethylxanthine, dexamethasone, and indo-
methacin [17]. In selected experiments, the media was supplemented
with L-()-norepinephrine-(þ) bitartrate (NE, Calbiochem, EMD Milli-
pore; Billerica, MA, USA), palmitate or oleate (SigmaeAldrich) conju-
gated with fatty acid-free BSA [18], 300 nM of the pan protein kinase C
(PKC) inhibitor phorbol 12-myristate 13-acetate (PMA) (LC Laboratories;
Woburn, MA, USA) [19] or 100
m
MH
2
O
2
(SigmaeAldrich) [20] and
replenished daily from d3ed7 of differentiation.
Heterologous protein expression in primary brown adipocytes was
achieved using recombinant adenovirus. The open reading frame of
Them1 was cloned into VQAd5-CMV (ViraQuest; North Liberty, IA, USA)
along with an N-terminal FLAG-tag downstream of the CMV promoter.
This was used to create the Them1 adenovirus (Ad-Them1) by stan-
dard techniques (ViraQuest). A recombinant GFP adenovirus (Ad-GFP,
ViraQuest), as well as Ad-Them2 [18] were used as controls. Re-
combinant adenoviruses were added to the medium for 24 h during d1
of differentiation.
2.7. O
2
consumption rates (OCR)
The O
2
consumption rate (OCR) of BAT were measured ex vivo using a
Clark electrode YSI 5300A Biological Oxygen Monitor System (YSI Life
Sciences; Yellow Springs, OH, USA). BAT was isolated from male 7e9
w-old mice after exposure to ambient temperatures of 22

Cor4

C
for 96 h. For duplicate measurements, BAT was separated into two
pieces, which were weighed separately. Immediately upon harvesting,
tissues were minced finely and suspended (50e70 mg BAT per 3 ml)
in unbuffered DMEM media containing 25 mM glucose, 10 mM sodium
pyruvate and 2 mM GlutaMAX (Life Technologies; Carlsbad, CA) in the
2 separate temperature-controlled chambers of the apparatus. Time-
dependent values of [O
2
] were determined for 5 min at 37

C. OCR
values (%/min) were determined by fitting to the equation [O
2
] (% initial
concentration) ¼100*e
OCR*t
. OCR for an individual BAT sample was
determined as the average of duplicate values. Values of OCR were
adjusted for differences in BAT weight by ANCOVA using VassarStats.
OCR values were measured in primary brown adipocytes using an
XF24 extracellular flux analyzer (Seahorse Bioscience; North Billerica,
MA, USA). Primary brown adipocytes were seeded at 25,000 cells/cm
2
in customized Seahorse 24-well plates (Seahorse Bioscience) and
differentiated as described above. Mature brown adipocytes were
incubated in the absence of CO
2
for 1 h at 37

C in KrebseHenseleit
buffer (pH 7.4) containing 0.45 g/L glucose, 111 mM NaCl, 4.7 mM
KCl, 2 mM MgSO
4
-7H
2
O, 1.2 mM Na
2
HPO
4
, 5 mM HEPES and 0.5 mM
carnitine (SigmaeAldrich). OCR values were measured before and
after the exposure of cells to NE, etomoxir (SigmaeAldrich), Atglistatin
(EMD Millipore) or palmitate (SigmaeAldrich) conjugated with fatty
acid-free BSA [18]. To determine the acute responses of OCR to NE,
etomoxir or Atglistatin, 300
m
l KrebseHenseleit buffer was added to
the medium (total 560
m
l). To determine the acute response of OCR to
palmitate, the culture medium was changed to 560
m
l KrebseHen-
seleit buffer. NE, FFA, Atglistatin and etomoxir were each dissolved in
75
m
l KrebseHenseleit buffer and added to the total 560
m
l of medium
from the injection ports of the Seahorse apparatus.
To test mitochondrial respiratory function, the culture medium was
changed to unbuffered DMEM media containing 25 mM glucose,
10 mM sodium pyruvate and 2 mM glutamax. The ATP synthase in-
hibitor, oligomycin, the mitochondrial uncoupler carbonyl cyanide-p-
trifluoromethoxyphenylhydrazone (FCCP), the electron transfer inhibi-
tor, rotenone and antimycin A (Seahorse) were sequentially added to
each well [18]. OCR values are represented either as pmol/min/mg of
protein or as the percentage of change when compared with basal OCR
values.
2.8. Rates of lipolysis
NE-induced lipolysis was measured in mature primary brown adipo-
cytes by measuring glycerol released into culture media using a free
glycerol determination kit (SigmaeAldrich) as described [12]. Briefly,
fully differentiated primary brown adipocytes were incubated for 1 h in
phenol red-free non-buffered DMEM (SigmaeAldrich) containing 2%
fatty acid-free bovine serum albumin (SigmaeAldrich). The media
were then replaced by fresh media plus 1
m
M NE. The media were
sampled periodically during a 3 h incubation period to determine
glycerol concentrations. Data were normalized to cellular protein
contents.
2.9. Fatty acid oxidation rates
Rates of fatty acid oxidation were determined in primary brown adi-
pocytes according to the conversion of [1e
14
C] palmitate (55 mCi/
mmol: American Radiolabeled Chemicals; St. Louis, MO, USA) into
14
C-
acid soluble metabolites (ASM) and -CO
2
[18]. Briefly, matured brown
adipocytes were incubated for 6 h in 2 ml of fresh culture medium with
10% FBS supplemented with 1 mM carnitine (SigmaeAldrich) and
0.25
m
Ci [1e
14
C] palmitate/ml plus 200
m
M palmitate conjugated with
Original article
342 MOLECULAR METABOLISM 5 (2016) 340e351 Ó2016 The Authors.Published by Elsevier GmbH.This is an open access article under theCC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
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fatty acid-free BSA. To determine ASM, the culture medium was mixed
in conical tubes with 200
m
l of 70% perchloric acid (Fisher Scientific;
Pittsburgh, PA, USA) with filter papers soaked in 2N NaOH placed in the
caps in order to trap
14
CeCO
2
. After incubation for 1 h on a horizontal
shaker, the filter papers were removed, and the acidified medium was
incubated overnight at 4

C and then centrifuged at 14,000 g for
20 min
14
C-labeled CO
2
in the filter paper and ASM in the supernatant
were dissolved in Ecoscint H (National Diagnostics; Atlanta, GA, USA)
and were counted using a LS6000IC liquid scintillation counter
(Beckman Coulter; Danvers, MA).
2.10. Immunoblot analysis
Tissue and cells were homogenized in RIPA buffer (1% Nonidet P-40,
0.1% sodium deoxycholate, 0.1% SDS, 150 mM NaCl, 50 mM Trise
HCl, 2 mM EDTA) for determination of protein expression by immu-
noblot analysis. Protein concentrations were determined spectropho-
tometrically using a Bio-Rad protein assay dye reagent, and equal
amounts of proteins separated by SDS-PAGE. Following electropho-
retic transfer to nitrocellulose membranes (GE Healthcare; Pittsburgh,
PA, USA), blots were probed with specific primary antibodies: Anti-
bodies to Them1 and
b
-Actin were as described previously [11]. An-
tibodies to Ucp1 and adipose triglyderide lipase (Atgl) were obtained
from Abcam (Cambridge, MA, USA). Antibodies to hormone sensitive
lipase (Hsl) and phospho- Hsl were from Cell Signaling Technology,
and antibodies to perilipin 1 (Plin1) from Fitzgerald Industries (Acton,
MA, USA). Primary antibodies were detected by enhanced chem-
iluminescence (SuperSignal West DURA, Fisher).
2.11. Statistics
Differences were evaluated using two-tailed unpaired Student’st-tests
when two groups were compared. Multiple group comparisons were
performed using two-way ANOVA. Differences were considered sig-
nificant for P<0.05.
3. RESULTS
3.1. Enhanced cold thermogenesis in Them1-deficient mice
We tested the regulatory role of Them1 in cold thermogenesis using
two experimental designs (Figure 1A,B, S1 and Table S1)inorderto
assess: 1) the acute response to exposures of mice to cold stress
(referred to here as “temperature-dynamic”), and 2) the equilibrated
response to prolonged exposures (referred to here as “temperature-
equilibrated”). We have previously demonstrated that these re-
sponses may differ substantially as mice acclimate to changes in
ambient temperature [12]. Daily energy expenditures were calculated
from hourly rates (Figure S1A and B), which were then adjusted for
lean body weights (Figure S1E and F). In Them1
/
mice, daily en-
ergy expenditure levels were increased at ambient temperatures of
22

C or lower under temperature-dynamic conditions (Figure 1A)
and at 4

C under temperature-equilibrated conditions (Figure 1B). At
4

C, Them1
/
mice had greater total daily energy expenditures
under both temperature-dynamic and temperature-equilibrated
conditions. Greater energy expenditures were also frequently
observed during the light phase. At the thermoneutral ambient
temperature of 30

C, increases in energy expenditure during the
light phase in Them1
/
mice most likely reflected incomplete
acclimation to the warm temperature since these mice were chron-
ically housed at 22

C[21].
For Them1
/
mice under temperature-dynamic conditions, significant
increases in adjusted mean hourly rates of energy expenditure were
observed at ambient temperatures of 22

C and below (Table S1). Under
temperature-equilibrated conditions, the increases in mean hourly rates
of energy expenditure were smaller in magnitude and did not them-
selves achieve statistical significance (Table S1). There were no sys-
tematic effects of Them1 expression on core body temperatures
(Figure S1C and D, and Table S1), food intake (Figure S1G and H)or
either daily or hourly physical activities (Figures S1IeLand Table S1).
Increased rates of energy expenditure in Them1
/
mice could have
resulted from changes in heat loss in response to alterations in vaso-
motor tone or skin lipid composition [22]. However, we did not observe
evidence of genotype-dependent differences in heat dissipation deter-
mined by surface infrared thermography as functions of ambient tem-
perature (Figure S1MeP).
Although Them1 is highly enriched in BAT, our use of a global Them1-
deficient mouse model leaves open the possibility that Them1 could be
acting to suppress energy expenditure by an alternative mechanism.
To address this issue, we measured rates of VO
2
in response to the
specific
b
3
-receptor agonist CL316,243 in mice housed at thermo-
neutrality (Figure 1CeE) [14,23]. Under these conditions, baseline
values of VO
2
were similar in Them1
þ/þ
and Them1
/
mice. Acute
b
3
-induced increases in VO
2
occurred similarly in both genotypes.
However, VO
2
values declined much more slowly in the absence of
Them1 expression (Figure 1C). Whereas this occurred in the absence
changes in Them1 mRNA (Figure 1D), the responses of thermogenic
genes to CL316,243 injection were largely preserved in Them1
/
mice (Figure 1E).
Histology of BAT sections revealed qualitative reductions in lipid droplet
size and abundance in Them1
/
mice, which were more pronounced
at 4

C(Figure 2A). This was validated by BAT triglyceride and FFA
concentrations, which were also reduced more markedly in Them1
/
mice at 4

C(Figure 2B). These changes were not accompanied by
alterations in BAT ultrastructure, as evidenced by electron microscopy,
which demonstrated typical appearing lipid droplets and mitochondria
in both genotypes (Figure 2A).
In plasma (Figure 2C), triglyceride concentrations were reduced by
housing mice at 4

C, but unaffected by Them1 expression. Plasma
FFA and glycerol concentrations in Them1
/
mice were increased at
22

C, but were not significantly higher at 4

C. These changes in
steady state plasma concentrations suggest greater triglyceride turn-
over due to increased demand for energy substrates by BAT. By
contrast, plasma glucose concentrations were decreased in the
absence of Them1 both at 22

C and 4

C. As previously reported [11],
Them1
/
mice exhibited more rapid clearance of exogenously
administered glucose and increased clearance rates of glucose during
insulin tolerance tests, suggesting that decreased plasma glucose
concentrations were attributable to greater glucose utilization, pre-
sumably by BAT of Them1
/
mice. Indeed, the absence of appre-
ciable changes in RER values (Table S1) supports the likelihood that
consumption of both fatty acids and glucose as energy substrates was
increased by the absence of Them1 in BAT.
3.2. Increased O
2
consumption in Them1-deficient BAT ex vivo
To further localize the effects of Them1 on energy expenditure, we
examined O
2
consumption in BAT ex vivo in response to a cold chal-
lenge. Compared with mice housed at 22

C, expression of Them1
(Figure 3A,B) and Ucp1 (Figure 3A,C) in BAT were both increased by
exposure to 4

C. More prolonged 96 h exposure to 4

C was asso-
ciated with further upregulation of Them1 protein, but with no further
increase in mRNA expression. There was no influence of Them1
expression on Ucp1 expression. In contrast to BAT, there was no in-
fluence of ambient temperature on Them1 expression in epididymal or
inguinal white adipose tissue (WAT) tissue or in liver (Figure S2A). Ucp1
MOLECULAR METABOLISM 5 (2016) 340e351 Ó2016 The Authors.Published by Elsevier GmbH. This is an open access articleunder the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
www.molecularmetabolism.com 343

expression in inguinal WAT was not influenced by absence of Them1
expression (Figure S2A).
Ratios of interscapular BAT to total body weight were increased by 96 h
exposure to 4

C, but this was not influenced by Them1 expression
(Figure 3D). The OCR in BAT was more rapid for mice housed at 4

C
than at 22

C(Figure 3E), and at each temperature, the absence of
Them1 expression increased the rate of decline. Because values of
OCR varied linearly as functions of excised BAT mass (Figure 3F), we
0
10
20
30
40
50
60
70
80
90
TLDTLDTLD
30 °C 22 °C 4 °C
1
1
1
2
2
2
2
2
3
3
3
3
3
4
4
4
5
6
7
8
0
10
20
30
40
50
60
70
80
90
TLDTLDTLDTLD
30 °C 22 °C 15 °C 4 °C
*
0
1
2
3
4
5
0 60 120 180 240 300 360
*
60
70
80
90
100
120 220 320
Daily
energy expenditure, kJ
AB
*
*
*
*
*
*
*
*
*
VO2(ml/hr/kg lean bw)
Percent max VO2
CL316,243
C
0
1
2
3Them1
Relative mRNA
expression
Them1:
D
PBS - 90 min
CL316,243 - 60 min
CL316,243 - 90 min
+/+ -/-
0
1
2
3
4
5
0
5
10
15
20
25
0
1
2
3Ucp1
0
2
4
6
8Pparγ
Pgc1αPparα
Relative mRNA
expression
†
††
†
Time (min)
+/+ -/- +/+ -/- +/+ -/- +/+ -/-
Them1:
E
***
***
******
*********
****
Figure 1: Them1 suppresses energy expenditure in response to thermal stress in mice. For mice (n ¼6/group) housed under (A) temperature-dynamic and (B) temperature-
equilibrated conditions, daily values of total (T) energy expenditure as well as the 12 h light (L) and dark (D) phases. For mice under temperature-equilibrated conditions (B), values
of energy expenditure represent averages of the 2 diurnal cycles. (C) Response to CL316,243 (0.5 mg/kg) of VO
2
values for mice housed at 30 C(n¼9/group). The inset panel
shows data normalized to maximum VO
2
values following CL316,243 injection, which did not differ between genotypes. Relative expression of mRNA in BAT of mice housed at
30 C(n¼3/group) for (D) Them1 and (E) thermogenic genes following injection of CL316,243 or PBS. Error bars represent SEM.
*
P<0.05, Them1
/
vs. Them1
þ/þ
mice;
y
P<0.05, CL316,243 vs. PBS.
Original article
344 MOLECULAR METABOLISM 5 (2016) 340e351 Ó2016 The Authors.Published by Elsevier GmbH.This is an open access article under theCC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
www.molecularmetabolism.com

evaluated genotype-dependent differences at each temperature by
adjusting for tissue mass. Independent of BAT mass, the absence of
Them1 was associated with a significant increase in these adjusted
OCR values in cold-challenged Them1
/
mice (Figure 3G). OCR
values were also increased at 22

C, but this did not achieve
significance.
To understand what might be driving enhanced thermogenesis in vivo
and elevated OCR of BAT, we examined gene expression of key BAT
genes that regulate differentiation, thermogenesis, fatty acid oxidation,
as well as compensation from other Acots (Table S2). Apart from
reduced upregulation of Acot8 at 4

CinThem1
/
mice, there were
no genotype-related differences suggesting non-transcriptional regu-
lation of thermogenesis by Them1. To determine if the enhanced
energy expenditure in BAT might be secondary to increased substrate
availability, we assessed protein phosphorylation of Hsl and Plin1.
Exposure to 4

C tended to promote phosphorylation of Hsl at each
major site, with tendencies towards reduced migration of Plin1 and
upregulation of Plin1 and Atgl expression (Figure S2B). However, there
were no differences attributable to Them1 expression.
3.3. Them1 reduces fatty acid oxidation in primary brown
adipocytes
Primary brown adipocytes were utilized to further probe regulatory
mechanisms of Them1. Unlike BAT, ex vivo differentiated primary brown
adipocytes expressed only low levels of Them1 (Figure S3A). In these
cells Ucp1 but not Them1 expression could be induced by pretreatment
of brown pre-adipocytes with NE (Figure S3A). The medium
concentration of FBS weakly influenced Them1 expression (Figure S3B),
which was not affected by exposure to palmitate, oleate, PMA and H
2
O
2
(Figure S3C). NE pretreatment increased basal OCR values, which varied
linearly as a function of Ucp1 expression levels (Figure S3D). To test
Them1 function, we chose 1
m
M NE to pretreat cells, which led only to a
25% decrease in the triglyceride contents of brown adipocytes irre-
spective of Them1 expression (Figure S3E, left panel), a slight decrease
in FFA concentration in the absence of Them1 expression (Figure S3E,
right panel) and to modest gene expression changes that were largely
independent of Them1 expression (Table S3).
Irrespective of Ucp1 induction by NE pretreatment, there were no
Them1-dependent differences in basal or NE-stimulated values of OCR,
which were likely due to the low levels of Them1 expression in this
system (Figure 4A,B). However, when Them1 expression was recon-
stituted to a similar level as expressed in wild type BAT using recom-
binant adenovirus at a multiplicity of infection(MOI) of 40 (Figure S3F and
G, and Table S3), suppression of OCR was observed: In the absence of NE
pretreatment, Them1 expression did not influence basal OCR values
(Figure 4C), but attenuated the increases observed following acute NE
stimulation. Following NE pretreatment, Them1 but not GFP expression
reduced both basal and NE-stimulated OCR values (Figure 4D). To
ensure specificity, we tested the effects of similarly reconstituting
Them2 (Figure S3H and I), the expression of which was more modestly
reduced in primary brown adipocytes (Figure S3F). Irrespective of pre-
treatment with NE, expression of Them2 did not influence either basal
OCR values or the response to NE stimulation in Them1
þ/þ
(Figure 4E,F)
or Them1
/
(Figure S3J and K) brown adipocytes.
C
[Glycerol], mg/dl
*
0.6
0.4
0.2
0
1.0
0.8
[Glucose], mg/dl
150
100
50
0
200
*
*†
†
[Triglycerides], mg/dl
30
20
10
0
50
40
[FFA], mM
0.6
0.4
0.2
0
1.0
0.8 *
Them1+/+
Them1-/-
4 °C22 °C
*
[Triglycerides], mg/g
150
100
50
0
200
[FFA], μmol/g
*
†
†
15
10
5
0
20
Them1
Them1
4 °C22
AB
Figure 2: Effects of Them1 expression on BAT structure and on tissue and plasma concentrations of energy substrates. (A) Representative images of BAT samples
subjected to hematoxylin and eosin staining and electron microscopy (abbreviations: LD, lipid droplet, M, mitochondria). (B) BAT concentrations of triglycerides and FFA. (C) Plasma
concentrations of triglycerides, FFA, glycerol and glucose (n ¼6/group). Error bars represent SEM.
*
P<0.05, Them1
/
vs. Them1
þ/þ
mice;
y
P<0.05, 4 C vs. 22 C.
MOLECULAR METABOLISM 5 (2016) 340e351 Ó2016 The Authors.Published by Elsevier GmbH. This is an open access articleunder the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
www.molecularmetabolism.com 345

3.4. Them1 suppresses oxidation of endogenous but not
exogenous fatty acids
In order to determine whether differences in OCR were attributable to
the availability of endogenous fatty acids, we treated primary brown
adipocytes with the carnitine palmitoyl transferase (CPT) 1 inhibitor,
etomoxir (Figure 5A). OCR values in cells lacking Them1 progressively
decreased to levels observed in Them1
/
brown adipocytes that were
reconstituted with Them1. In the presence of etomoxir, NE stimulation
only modestly increased OCR values, irrespective of Them1 expres-
sion. To further examine whether higher basal OCR values observed in
the absence of Them1 were attributable to oxidation of lipid droplet-
derived fatty acids, we utilized the Atgl inhibitor Atglistatin
(Figure 5B). Addition of Atglistatin also reduced OCR values in GFP-
expressing Them1
/
brown adipocytes to values observed in
Them1-expressing cells, and eliminated the differences in response to
NE stimulation. This was not attributable to lipid availability because in
absence of NE pretreatment, reconstitution of Them1 expression
influenced neither triglyceride nor FFA concentrations (Figure 5C).
Consistent with reduced triglyceride concentrations due to NE pre-
treatment, rates of glycerol release were also reduced in response to
acute NE stimulation (Figure 5D). However, Them1 expression did not
influence glycerol release under either experimental condition, sug-
gesting that the primary effect of Them1 was on basal OCR. We further
examined phosphorylation of Hsl and Plin1, as well as expression of
Atgl (Figure S4A), but observed no genotype-related differences with or
without NE pretreatment.
In response to exogenous palmitate, OCR values did not differ in GFP-
and Them1-reconstituted cells (Figure 5E). Moreover, oxidation of
exogenous
14
C-palmitate into
14
C-ASM and
14
C-CO
2
was unchanged in
the absence of Them1 expression (inset to Figure 5E). Similarly,
expression of Them2 had no effect on the oxidation of exogenous fatty
acids (Figure S4B). To examine the influence of Them1 expression on
cellular mitochondrial function, primary brown adipocytes were
sequentially exposed to oligomycin, FCCP, and antimycin plus rote-
none, which assess ATP-linked O
2
consumption, maximum OCR, and
non-mitochondrial OCR, respectively [12]. There were no differences in
cellular bioenergetic parameters (Figure 5F), indicating that Them1
expression did not influence intrinsic mitochondrial function. In
keeping with previous demonstrations that endogenous Them2
expression does not alter mitochondrial function in brown adipocytes
[12], there were no effects of heterologous Them2 expression
(Figure S4C).
4. DISCUSSION
The current findings support a function of Them1 in suppressing cold-
induced thermogenesis in BAT, and the data are consistent with a
model in which Them1 limits basal hydrolysis of long chain fatty acyl-
CoAs, reducing mitochondrial uptake and
b
-oxidation (Figure 6).
In the setting of cold stress, Them1 reduced energy expenditure, and
the magnitude of this effect did not diminish over time (i.e. the effect
was observed under both temperature-dynamic and temperature-
equilibrated conditions). Because Them1 expression was minimal in
WAT and did not influence Ucp1 expression, browning of WAT might
have been expected to compensate for Them1-mediated suppression
of BAT thermogenesis [24,25]. However, the inhibitory effects of
†
Relative protein expression
Them1
2
1
0
4
3
†
22°C 4°C
DE
120
80
40
0
20
60
100
012345
[O2in solution], % max
Time, min
Them1
Them1
BAT, % of BW (x10)
6
4
2
0
8
††
Them1
Them1
F
OCR, % O2/min
BAT weight, mg
60 80 100 120 140 160
0.3
0.2
0.1
0
G
Them1
β-Actin
Them1+/+ Them1-/-
22°C 4°C
Ucp1
-24 h 96 h -24 h 96 h
AB
Relative mRNA expression
††
Them1
3
2
1
0
4
C
Ucp1
2
1
0
3
††
Relative protein expression
BAT
Adjusted OCR, % O2/min
Ambient temperature, °C
0.30
0.25
0.15
0.20
30 20 10 0
*
†
†
Them1
Them1
-
422
24 96
°C
h
422 °C422 °C
Figure 3: Ambient temperature regulates Them1 expression and OCR in BAT. (A) Them1 and Ucp1 expression in BAT homogenates. (B) Expression levels of Them1 protein relative
to
b
-Actin and mRNA (n ¼4e6/group). (C) Ucp1 protein relative to
b
-Actin. Freshly isolated interscapular BAT from 7 to 9 w-old mice (n ¼5e9/group) analyzed for (D) BAT weight
normalized by body weight, (E) O
2
concentrations expressed as % of the initial value and fit to the equation [O
2
]¼100*e
OCR*t
and (F) dependence of OCR on BAT mass. (G) OCR values
were adjusted for BAT mass by ANCOVA at 22 C and 4 C. Error bars represent SEM.
*
P<0.05, Them1
/
vs. Them1
þ/þ
mice;
y
P<0.05, 22 C vs. 4 C.
Original article
346 MOLECULAR METABOLISM 5 (2016) 340e351 Ó2016 The Authors.Published by Elsevier GmbH.This is an open access article under theCC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
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Them1 on energy expenditure were unabated after 96 h cold exposure,
presumably reflecting the increase in Them1 expression in BAT that
occurred during this period.
Consistent with an effect of Them1 on basal thermogenesis, the
increased energy expenditure in Them1
/
mice at reduced ambient
temperatures was most evident during the light phase. These in-
creases were not explained by increases in physical activity or core
body temperatures. Consistent with an effect restricted to non-
shivering thermogenesis, increases in daily energy expenditures
were not detected during the dark phase, when physical activity and
food intake were maximal. However, we did observe higher values of
hourly energy expenditures during the dark phase in Them1
/
mice,
suggesting that basal differences were nevertheless still present in the
dark.
A suppressive effect of Them1 on cold-induced thermogenesis was
supported by in vivo,ex vivo and in vitro experiments. When taken
together with increased OCR values in BAT isolated from Them1
/
mice subjected to cold stress, these observations most likely explain
reduced lipid droplet abundance and decreased tissue triglyceride and
FFA concentrations. The elevated plasma concentrations of FFA and
glycerol and reduced glucose concentrations, as well as increased
insulin sensitivity of Them1
/
mice are consistent with increased
010020 40 60 80 010020 40 60 80
7
6
5
4
3
2
AUC (x105)
6
4
2
0
Basal
NE
AUC (x105)
6
4
2
0
Basal
NE
7
6
5
4
3
2
OCR, pmol/min/mg (x104)
Them1
Them1
-+
NE pretreatment
NE NE
AUC (x105)
6
4
2
0
Basal
NE
*
010020 40 60 80
010020 40 60 80
7
6
5
4
3
2
OCR, pmol/min/mg (x104)
*
*
AUC (x105)
6
4
2
0
Basal
NE
*
*
****
7
6
5
4
3
2
NE NE
Ad-GFP
Ad-Them1
-+
NE pretreatment
Time, min
010020 40 60 80
7
6
5
4
3
2
AUC (x105)
6
4
2
0
Basal
NE
A
C
E
7
6
5
4
3
2
Time, min
010020 40 60 80
OCR, pmol/min/mg (x104)
AUC (x105)
6
4
2
0
Basal
NE
NE NE
Ad-GFP
Ad-Them2
-+
NE pretreatment
B
D
F
**
******
*
Figure 4: Them1 suppresses fatty acid oxidation in primary brown adipocytes. Response of OCR values (n ¼10) to stimulation with 1
m
M NE for (A,B) Them1
þ/þ
and
Them1
/
brown adipocytes (data are representative of 2 independent experiments), (C,D) Them1
/
brown adipocytes reconstituted with Them1 using Ad-Them1 (MOI 40) (data
are representative of 3 independent experiments) and (E,F) Them1
þ/þ
brown adipocytes reconstituted with Them2 using Ad-Them2 (MOI 40). In experiments using adenovirus, Ad-
GFP (MOI 40) served as the control. The bar graphs in each panel provide basal and NE-stimulated AUC values. Error bars represent SEM.
*
P<0.05, Ad-Them1 vs. Ad-GFP.
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www.molecularmetabolism.com 347

caloric demands of BAT. Presumably because increased glucose up-
take and oxidation occur in response to BAT activation [26],Them1
/
mice did not exhibit changes in RER values. Considering that 60% or
more of mouse energy expenditure in the cold is attributable to BAT
thermogenesis [27], the 15% increase in OCR in isolated BAT from cold
exposed Them1
/
mice should be sufficient to account for the w7%
increases in cumulative daily energy expenditure observed in the intact
animal. Although we did not detect an increase in body temperatures of
Them1
/
mice in the setting of their higher metabolic rates, our
thermography data do not support a primary thermoregulatory
phenotype whereby increased heat loss served to promote accelerated
metabolic fluxes in the cold [22]. Since
b
3
-agonist-induced increases
in VO
2
at thermoneutrality are primarily attributable to activation of BAT
[14,21], the much slower decline in VO
2
values for Them1
/
mice
following CL316,243 injection that occurred in the absence of altered
thermogenic gene expression, support a direct role for Them1 in
suppressing BAT thermogenesis. Although we cannot exclude a role
for Them1 in skeletal muscle fatty acid and glucose metabolism,
Them1 is expressed a very low levels in skeletal muscle compared to
BAT [11]. However, a BAT-specificThem1
/
mouse would be
required to distinguish these possibilities with certainty.
Studies in primary BAT cells provided additional insights into a
mechanistic role for Them1 in reducing oxidation of endogenous fatty
acids. Increases in OCR attributable to the absence of Them1 were
abrogated by inhibiting CPT1, which is rate-limiting for the mito-
chondrial uptake of fatty acids, or Atgl, which is the rate-limiting
enzyme for lipid-droplet triglyceride hydrolysis [28]. Although its pre-
cise subcellular localization remains unknown, Them1 in BAT of mice
housed at room temperature was, upon subcellular fractionation,
largely concentrated in the endoplasmic reticulum (ER) [9,11], which
plays critical roles in the metabolism of lipid droplets [29]. Recent
studies have suggested that Them1 translocates to the lipid droplet in
OCR, pmol/min/mg (x104)
*
* *
Time, min
0 100 20 40 60 80
NE Atglistatin
[Glycerol], mg/g protein
6
4
2
0
60 120
180
Time, min
0
E F
A B
D
C
Ad-GFP
Ad-Them1
- +
NE pretreatment
Time, min
0 100 20 40 60 80
OCR, pmol/min/mg (x104)
NE
* *
*
6
5
4
3
Etomoxir
0.6
0.4
0.2
0
†
[Triglycerides], mg/mg protein
0.6
0.4
0.2
0
[FFA], mmol/mg protein
†
Ad-GFP
Ad-Them1
- +
- +
NE pretreatment
200
150
100
50
0
Time, min
0 100 20 40 60 80
OCR, % baseline
ASM CO2
[C] Palmitate
Oxidation
(mol/mg/min)
8
4
2
0
6
Palmitate
OCR, pmol/min/mg (x104)
Time, min
0 100 20 40 60 80
6
4
2
0
Oligomycin Antimycin/Rotenone FCCP
* *
* *
6
5
4
3
Figure 5: Them1 suppresses Atgl-dependent oxidation of endogenous fatty acids. Primary brown adipocytes from BAT of Them1
/
mice were exposed (MOI 40) to
recombinant Ad-Them1 or Ad-GFP and pretreated with NE. Response of OCR values (n ¼10) to the sequential addition to the media of (A) 200
m
M etomoxir and 1
m
M NE or (B)
40
m
M Atglistatin [38] and 1
m
M NE to the media (data are representative of 2 independent experiments). (C) Influence of NE pretreatment on triglyceride and FFA concentrations in
primary brown adipocytes (n ¼5e8/group). (D) Rates of triglyceride lipolysis (n ¼3) following the addition of 1
m
M NE (data representative of 2 independent experiments). Data for
Ad-GFP in the absence of NE pretreatment are not visualized because they fall behind the data points for Ad-Them1. (E) Response of OCR values to 300
m
M palmitate as indicated
by the arrow (n ¼10). Inset: Oxidation rates (n ¼3) of 200
m
M[1e
14
C] palmitate. (F) Response of OCR values (n ¼10) to the sequential addition of oligomycin (2
m
M), FCCP
(1
m
M), and rotenone (1
m
M) plus antimycin A (1
m
M) [12]. Error bars represent SEM.
*
P<0.05, Ad-Them1 vs. Ad-GFP;
y
P<0.05, with 1
m
M NE pretreatment vs no NE
pretreatment.
Original article
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the setting of lipolytic stimulation. A proteomic analysis of BAT lipid
droplets isolated from mice exposed to the 4

C compared with mice
housed at room temperature revealed the association of Them1 with
lipid droplets, along with a 2.5-fold enrichment following cold exposure
[30]. The same analysis revealed cold-mediated enrichment of BAT
lipid droplets with both HSL and ATGL. We visualized cold-induced
relocation of Them1 to BAT lipid droplets by colocalization with
Plin1, and this was validated with immunoblot analysis of isolated lipid
droplets [31]. As a result, Them1 may be well positioned to regulate
lipolysis by generating FFA in close proximity to lipid droplets under
lipolytic conditions.
Whereas Atgl activity is only modestly suppressed by FFA, fatty acyl-
CoAs are potent non-competitive inhibitors [32]. This suggests that
Atgl may be inhibited when Them1-generated FFA are converted
locally with fatty acyl-CoAs. Indeed, considerable evidence suggests
that FFA esterification to CoASH is compartmentalized by Acsl isoforms
that are regionally distributed within cells [33]. Whereas Acsl1 pro-
motes mitochondrial fatty acyl-CoA uptake [34], a distinct Acsl (rep-
resented as Acslx in Figure 6) presumably generates fatty acyl-CoAs
that both inhibit Atgl and serve as substrates for lipid droplet tri-
glycerides. Because this model depends in part on the use of phar-
macologic inhibitors, it should be strengthened by corresponding
genetic gain- and loss-of function studies, as well as to the Acslx once
it is identified.
Because the enzymatic activity of Them1 contributes to the overall
balance of FFA and fatty acyl-CoAs in BAT [9,11], another potential
mechanism by which it could regulate thermogenesis in BAT is via
peroxisome proliferator-activated receptor (Ppar) activation [1]. This
was unlikely because we did not observe Them1-dependent changes
in mRNA levels of Ppar targets or of other key thermogenic genes,
including Acsl1 and Fabp3, which function to promote fatty acid
oxidation within BAT [34,35]. The absence of changes in expression
levels of a broad panel of regulatory genes supports a non-
transcriptional mechanism whereby Them1 suppresses cold
thermogenesis.
The absence of Them1 upregulation by
b
3
-adrenergic stimulation
in vivo or by NE pretreatment of primary brown adipocytes suggests
that the transcriptional regulation of Them1 by ambient temperature
may occur by a mechanism distinct from the cold-driven transcrip-
tional thermogenic program of BAT [36]. However, Them1 expression
was not restored in response to exogenous FFA, serum or reactive
oxygen species in the form of H
2
O
2
. Protein kinase C (PKC)
b
can
suppress thermogenesis [37], but a PKC activator did not reconstitute
Them1 expression. As evidenced by an increase in protein expression
following prolonged cold exposure that was unaccompanied by in-
creases in mRNA levels, Them1 upregulation also appears to involve
post-translational effects. The mechanisms by which cold exposure
upregulates Them1 expression require further investigation.
Taken together, our findings suggest the presence of an active feed-
back mechanism that limits cold thermogenesis in BAT even in the
setting of a prolonged cold stress. This likely underscores the survival
value of energy conservation, such that the calorie intensive process of
non-shivering thermogenesis is rapidly suppressed when no longer
necessary. Because it reduces thermogenesis in proportion to its
expression level and without broadly altering gene expression, Them1
may represent an attractive target for management of obesity and
related metabolic disorders.
AUTHOR CONTRIBUTIONS
Author contributions: K.O. and D.E.C. designed research; K.O., K.B.L.,
Y.Z., Y.L., T.I.K., C.O. and S.J.H. performed research; K.O., K.B.L.,
S.J.H., T.I.K., R.A.B., A.S.B. and D.E.C. analyzed data; and K.O., A.S.B.
and D.E.C. wrote the paper.
Plasma
Cytosol
β3-AR
Norepinephrine COLD EXPOSURE
PKA
Hsl
Plin1
Atgl
P
Lipid
Droplet
Fatty acid
Acsl1
Them1
Mitochondria
β-oxidation
of fatty acids
THERMOGENESIS
Cpt1
UCP1
ETC
H+
H+
NADH
Brown adipocyte
Fatty acyl-CoA
Acslx
Fatty acyl-CoA
Figure 6: Schematic model of suppression of cold thermogenesis by Them1. In response to cold exposure, NE release from sympathetic neurons activates the
b
3-adrenergic
receptor (
b
3
-AR), leading to activation of PKA. PKA in turn stimulates lipolysis by phosphorylation of Plin1, which leads to activation of Atgl, the rate-limiting step in lipid droplet
triglyceride hydrolysis. PKA also phosphorylates and activates Hsl. FFA liberated as a result of lipolysis are the activated by Acsl1 and taken up into mitochondrial by the activity of
carnitine palmitoyl transferase 1 (Cpt1). The proton-gradient generated by the activity of the electron transport chain following
b
-oxidation of fatty acids is uncoupled from ATP
synthesis by Ucp1. Them1 opposes the activity of Acsl1, generating FFA that are then converted by an as yet identified Acsl (i.e. Acslx), which suppress activity of Atgl and may be
reassembled into triglycerides for lipid droplet storage.
MOLECULAR METABOLISM 5 (2016) 340e351 Ó2016 The Authors.Published by Elsevier GmbH. This is an open access articleunder the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
www.molecularmetabolism.com 349

ACKNOWLEDGMENTS
This work was supported by U.S. National Institutes of Health grants R01 DK056626
and R37 DK048873 (to D.E.C.), R01 DK103046 (to D.E.C. and S.J.H.) the Harvard
Digestive Diseases Center (P30 DK034854) and the Harvard Catalyst, support by the
National Center for Advancing Translational Sciences (UL1 TR001102).
CONFLICT OF INTEREST
None declared.
APPENDIX A. SUPPLEMENTARY DATA
Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.
molmet.2016.02.002.
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