Deficiency of TNFα converting enzyme (TACE/ADAM17) causes a lean, hypermetabolic phenotype in mice

Article (PDF Available)inEndocrinology 149(12):6053-64 · September 2008with78 Reads
DOI: 10.1210/en.2008-0775 · Source: PubMed
Abstract
Energy homeostasis involves central nervous system integration of afferent inputs that coordinately regulate food intake and energy expenditure. Here, we report that adult homozygous TNFalpha converting enzyme (TACE)-deficient mice exhibit one of the most dramatic examples of hypermetabolism yet reported in a rodent system. Because this effect is not matched by increased food intake, mice lacking TACE exhibit a lean phenotype. In the hypothalamus of these mice, neurons in the arcuate nucleus exhibit intact responses to reduced fat mass and low circulating leptin levels, suggesting that defects in other components of the energy homeostasis system explain the phenotype of Tace(DeltaZn/DeltaZn) mice. Elevated levels of uncoupling protein-1 in brown adipose tissue from Tace(DeltaZn/DeltaZn) mice when compared with weight-matched controls suggest that deficient TACE activity is linked to increased sympathetic outflow. These findings collectively identify a novel and potentially important role for TACE in energy homeostasis.
Deficiency of TNF
Converting Enzyme (TACE/ADAM17)
Causes a Lean, Hypermetabolic Phenotype in Mice
Richard W. Gelling, Wenbo Yan, Salwa Al-Noori, Aaron Pardini, Gregory J. Morton, Kayoko Ogimoto,
Michael W. Schwartz, and Peter J. Dempsey
Division of Metabolism, Endocrinology, and Nutrition (R.W.G., A.P., G.J.M., K.O., M.W.S., P.J.D.), Department of Medicine,
University of Washington, Seattle, Washington 98195; Pacific Northwest Research Institute (S.A.-N., P.J.D.), Seattle,
Washington 98122; and Departments of Molecular and Integrative Physiology and Pediatrics (W.Y., P.J.D.), University of
Michigan, Ann Arbor, Michigan 48109
Energy homeostasis involves central nervous system integra-
tion of afferent inputs that coordinately regulate food intake
and energy expenditure. Here, we report that adult homozy-
gous TNF
converting enzyme (TACE)-deficient mice exhibit
one of the most dramatic examples of hypermetabolism yet
reported in a rodent system. Because this effect is not matched
by increased food intake, mice lacking TACE exhibit a lean
phenotype. In the hypothalamus of these mice, neurons in the
arcuate nucleus exhibit intact responses to reduced fat mass
and low circulating leptin levels, suggesting that defects in
other components of the energy homeostasis system explain
the phenotype of Tace
Zn/Zn
mice. Elevated levels of uncou
-
pling protein-1 in brown adipose tissue from Tace
Zn/Zn
mice
when compared with weight-matched controls suggest that
deficient TACE activity is linked to increased sympathetic
outflow. These findings collectively identify a novel and
potentially important role for TACE in energy homeostasis.
(Endocrinology 149: 6053–6064, 2008)
T
NF
CONVERTING ENZYME (TACE/ADAM17) is a
prototypic member of the disintegrin-metalloprotease
(ADAM) family that is involved in proteolytic ectodomain
shedding (1–3). TACE was originally identified as the met-
alloprotease responsible for ectodomain cleavage of the
membrane-bound TNF
precursor to generate the soluble
cytokine, TNF
(4, 5). Since then, TACE has been shown to
possess broad sheddase activity that is required for the ef-
ficient ectodomain cleavage of a variety of type I and II
transmembrane proteins including growth factors, cyto-
kines, cytokine receptors, and cell adhesion molecules in vitro
(1–3). However, despite the identification of this large num-
ber of diverse TACE substrates by in vitro analysis, the phys-
iological importance of these shedding events in vivo, for the
most part, has not been determined.
Recently several insights into TACE function in vivo have
been obtained through analysis of mice lacking functional
TACE (Tace
Zn/Zn
). Tace
Zn/Zn
mice have a targeted deletion
of exon 11 that encodes the catalytic active site of the TACE
metalloprotease domain, resulting in a lack of enzymatic
activity (6). These animals display substantial perinatal le-
thality with several phenotypic defects characteristic of
epidermal growth factor (EGF) receptor (EGFR)-deficient
(Egfr
/
) mice (7–9). These include the open-eye phenotype
and altered eyelid, hair and whisker development of TGF
-
deficient mice (10, 11), the aberrant heart valve development
characteristic of heparin binding EGF-like growth factor
(HB-EGF)-null (12, 13), and defects in mammary morpho-
genesis observed in amphiregulin-deficient mice (14). Based
on these observations, TACE is hypothesized to be an es-
sential sheddase for the activation of at least three EGFR
ligands during development. More recently the in vivo anal-
ysis of radiation chimeric mice reconstituted with Tace
Zn/Zn
hematopoietic cells has demonstrated an important role
for TACE in shedding of several leukocyte substrates in-
cluding TNF
, TNF receptor (TNFR)-1, TNFR2, and l-
selectin (15, 16).
Modulation of TACE expression and activity can also alter
substrate shedding and therefore affect downstream signal-
ing and cellular responses (17–19). For example, when het-
erozygous Tace
/Zn
mice, which are viable and fertile (6), are
made homozygous for an impaired EGFR allele (wa-2), more
animals are born with the open eye phenotype, suggesting
that in the haplo-insufficient state, TACE activity is limiting
for effective EGFR signaling in vivo (19).
Recently, we characterized a population of homozygous
Tace
Zn/Zn
null mice that survive to adulthood and demon
-
strated that non-cell autonomous TACE expression was re-
quired for T cell development and peripheral B cell matu-
ration in vivo (20). The impaired B cell follicle organization
and germinal center formation in secondary lymphoid or-
gans observed in Tace
Zn/Zn
mice displayed features that
overlap with those found in TNF
-deficient mice, which
suggests a physiological role for TACE in activating TNF
signaling (20 –23). Another feature of the adult Tace
Zn/Zn
First Published Online August 7, 2008
Abbreviations: ADAM, A disintegrin and metalloprotease-17; ARC,
arcuate nucleus; BAT, brown adipose tissue; CNS, central nervous sys-
tem; E, embryonic day; EGF, epidermal growth factor; EGFR, EGF re-
ceptor; HB-EGF, heparin binding EGF-like growth factor; H&E, hema-
toxylin and eosin; MEF, mouse embryonic fibroblast; NPY, neuropeptide
Y; PREF-1, preadipocyte factor 1; QMR, quantitative magnetic reso-
nance; RER, respiratory quotient; SNS, sympathetic nervous system;
TACE, TNF
converting enzyme; TNFR, TNF receptor; UCP, uncou-
pling protein; VO
2
, rate of oxygen uptake; WAT, white adipose tissue;
WT, wild type; WT-CR, WT littermates calorically restricted.
Endocrinology is published monthly by The Endocrine Society (http://
www.endo-society.org), the foremost professional society serving the
endocrine community.
0013-7227/08/$15.00/0 Endocrinology 149(12):6053–6064
Printed in U.S.A. Copyright © 2008 by The Endocrine Society
doi: 10.1210/en.2008-0775
6053
mice was a dramatic reduction in body weight. However,
TNF
signaling is unlikely to play a major role in this phe-
notype because neither TNF
- nor TNFR-deficient mice
show significant changes in body weight when fed a stan-
dard chow diet (21–23).
The generation of adult Tace
Zn/Zn
mice provides a unique
opportunity to further investigate the role of TACE signaling
in adult physiology. The aim of the present study was to
characterize in detail the reduced body weight phenotype of
these mice. We found that adult Tace
Zn/Zn
null mice have
a lean, profoundly hypermetabolic phenotype. A central fea-
ture of this phenotype is a dramatic increase of metabolic rate
that is not due to increases of physical activity, body tem-
perature, or thyroid function. Adult Tace
Zn/Zn
mice also
display appropriate responses of arcuate nucleus (ARC) neu-
rons to reduced fat mass and leptin levels but, despite this,
fail to mount the expected decrease in energy expenditure or
increased food consumption normally observed in this set-
ting. Comparison with wild-type littermates that were ca-
lorically restricted to achieve a fat mass similar to that of
Tace
Zn/Zn
mice revealed disturbances of several down
-
stream signaling events within both the central nervous sys-
tem (CNS) and periphery of Tace
Zn/Zn
mice. Collectively,
these data suggest a previously unrecognized role for TACE
in the control of energy homeostasis.
Materials and Methods
Mice
All experiments were conducted using 8- to 16-wk-old male Tace
Zn/Zn
mice and control age-matched wild-type littermates unless otherwise
stated. Mice were maintained in a temperature-controlled room with a
12-h light, 12-h dark cycle and were provided with ad libitum access to
standard laboratory chow and water. All procedures were approved by
the Animal Care and Use Committees (University of Washington and
Pacific Northwest Research Institute) in accordance with National In-
stitutes of Health Guidelines for the Care and Use of Animals.
Body weight and length measurements
Cumulative body weight was measured once per week in group-
housed mice from 4 to 12 wk of age. Snout-anus length was measured
on 13-wk-old anesthetized animals.
Adipocyte differentiation
Mouse embryonic fibroblasts (MEFs) were derived from 14.5-d-old
wild-type, Tace
Zn/
and Tace
Zn/Zn
embryos. Early passage cells (pas
-
sage 3 or earlier) were used for differentiation of primary cells. Adipo-
cyte differentiation was initiated 2 d after cells reached postconfluence
by the addition of differentiation medium 10% fetal bovine serum-
DMEM containing 5
g/ml insulin, 1
m dexamethasone, 0.5 mm
3-isobutyl-1-methylxanthine, and 10
m troglitazone (24). After 2 d,
differentiation mixture was removed and culture was continued in 10%
fetal bovine serum-DMEM containing insulin and troglitazone. Fresh
media was added every 2–3 d. At d 7–10, cells were with fixed 4%
paraformaldehyde and stained with Oil Red O. For quantization, Oil red
O staining was eluted from cells with 0.5 ml 60% isopropanol and
absorbance read at 540 nm. All experiments were performed in triplicate.
Body composition analysis
In vivo body composition analysis of lean mass, fat mass, and water
content from conscious, immobilized mice was performed by quanti-
tative magnetic resonance (QMR) (EchoMRI whole-body composition
analyzer; Echo Medical Systems, Houston, TX) (25, 26).
Indirect calorimetry
Mice were individually housed and acclimated to the calorimeter
cages for 1 d before 1–3 d of data collection of gas exchanges and food
intake. Indirect calorimetry was performed with a computer-controlled
open circuit calorimetry system (Oxymax; Columbus Instruments Co.,
Columbus, OH) comprised of four respiratory chambers equipped with
a stainless steel elevated wire floor, water bottle, and food tray connected
to a balance. Oxygen consumption and CO
2
production were measured
for each mouse at 6-min intervals, and outdoor air reference values were
determined after every 10 measurements. Instrument settings were: gas
flow rate 0.5 liters/min, settle time 240 sec, measure time 60 sec.
Gas sensors were calibrated daily with primary gas standards containing
known concentrations of O
2
,CO
2
, and N
2
(Tech Air). A mass flow meter
was used to measure and control air flow. Oxygen was measured by an
electrochemical sensor using a limited diffusion metal air battery. CO
2
was
measured with a spectrophotometric sensor. Respiratory quotient (RER)
was calculated as the ratio of CO
2
production (liters) over O
2
consumption
(liters). Energy expenditure was calculated by the equation: energy expen-
diture (3.815 1.232 VCO
2
/VO
2
) O
2
consumption (rate of oxygen
uptake; VO
2
)
.
For thermoneutral conditions, calorimetry measurements
were performed as described above except that the ambient temperature of
the facility housing the calorimetry system was raised to 31 C.
Locomotor activity and feeding behavior
Locomotor activity, feeding, and drinking behavior were monitored
continuously during all indirect calorimetry experiments. Locomotor
activity was evaluated using an Opto-Varimetrix-3 sensor system (Co-
lumbus Instruments). Consecutive adjacent infrared beam breaks were
scored as an ambulatory count. Cumulative ambulatory activity counts
were recorded every hour for 24 h. The feeding behavior (i.e. frequency/
timing/duration and the amount of food/water consumed) was quan-
tified using feed-scale (mass) measurements. In separate experiments,
daily food intake was monitored by weighing food hoppers.
Caloric-restriction and fasting studies
Wild-type age- and sex-matched littermates were calorically re-
stricted to a similar relative fat mass as Tace
Zn/Zn
null mice. This was
achieved by providing mice with only 70% of their normal food intake
for the light and dark cycles at 0900 and 1700 h, respectively. Body
composition was monitored daily by QMR. Upon reaching the appro-
priate reduced fat mass, mice were examined by indirect calorimetry and
for locomotor activity, food intake, and water consumption. Caloric
restriction of mice was continued during indirect calorimetry experi-
ments. In separate experiments, wild-type age- and sex-matched litter-
mates were fasted for 24 48 h. Ad libitum wild-type littermates were
used as controls for both experimental conditions.
Plasma measurements
Leptin and corticosterone plasma levels were determined using spe-
cific RIAs that were performed by the Vanderbilt Mouse Metabolic
Phenotyping Center. Free T
4
plasma levels were determined by com
-
petitive enzyme immunoassay (Leinco Technologies, St. Louis, MO).
Tissue isolation
Mice were anesthetized and blood collected by cardiac puncture. All
tissues were rapidly dissected and frozen for subsequent biochemical
analysis or mRNA determination as previously described (27). For the
hypothalamus, a rectangular region of mediobasal hypothalamus (de-
fined caudally by the mamillary bodies; rostrally by the optic chiasm;
laterally by the optic tract; and superiorly by the apex of the hypotha-
lamic third ventricle) was isolated (27).
mRNA analysis
RNA from tissue was isolated and underwent RT-PCR quantification
as previously described (27). Total RNA was extracted from tissue using
RNAzol B according to the manufacturers’ instructions (Tel-Test, Inc.,
Friendswood, TX). RNA was calculated by spectrophotometry at 260
nm, and 1
g RNA was reverse transcribed with 10 U avian myelo-
6054 Endocrinology, December 2008, 149(12):6053– 6064 Gelling et al. TACE Regulation of Energy Expenditure
blastosis virus reverse transcriptase (Promega Corp., Madison, WI). PCR
was performed on a LightCycler (Roche Molecular Biochemicals, Indi-
anapolis, IN) using a 50-ng sample of cDNA template added to the
commercially available LightCycler PCR master mix (FastStart DNA
Master SYBR Green I; Roche Molecular Biochemicals). Primers were
designed to span an exon/intron boundary and optimized for mRNA
encoding Npy, Agrp, Pomc, Mch, Crh, Trh, and Gapdh. Primer sequences
can be obtained on request. Expression levels of individual hypotha-
lamic mRNAs were normalized to glyceraldehyde-3-phosphate dehy-
drogenase mRNA content and nontemplate controls were incorporated
into each PCR run.
Determination of cellularity
Four random digital images were captured from individual hema-
toxylin and eosin (H&E) stained sections of white adipose tissue (WAT)
and brown adipose tissue (BAT) obtained from four to five different
female mice of each genotype. The number nuclei were determined in
three fields for each fat pad using the National Institutes of Health
ImageJ software (Bethesda, MD). Data are presented as mean sem.
Western blotting
BAT or gonadal WAT was dissected and homogenized. Protein con-
tent was determined and Western blots using 20
g of total protein were
performed using goat antimouse uncoupling protein (UCP)-1 antibody
(1:1000) and goat anti-actin antibody (1:500) (Santa Cruz Biotechnology,
Santa Cruz, CA) as previously described (28).
Histology and immunohistochemistry
All tissues were immersion fixed with 4% paraformaldehyde unless
otherwise stated. Tissues were processed, paraffin embedded, and tissue
sections (8
m) prepared by routine procedures. H&E staining were
performed by standard histological methods.
Statistical analysis
All results are expressed as mean sem. A two-sample unpaired t
test was used for two-group comparisons. A one-way ANOVA with a
Newman-Keuls ad-hoc test was used to compare means between mul-
tiple groups. In all instances, P 0.05 was considered significant.
Results
Generation of Tace
Zn/Zn
mice that survive to adulthood
In the current study, all analyses were performed using
Tace
Zn/Zn
mice and littermate controls on a mixed (C57BL/
6 129) background (20) (supplemental methods, published
as supplemental data on The Endocrine Society’s Journals
Online web site at http://endo.endojournals.org). Embryos
derived from heterozygous Tace
Zn/
matings were exam
-
ined at embryonic days (E) 12.5, 14.5, and 17.5. Consistent
with the findings of Peschon et al. (6), the three expected
genotypes were found at the normal Mendelian ratio until
E17.5, when a reduction in the appearance of Tace
Zn/Zn
embryos was observed (supplemental Table 1). Similarly, a
large proportion of Tace
Zn/Zn
pups died in the first 24 h after
birth and during the first weeks of postnatal life. However,
in contrast to the original report by Peschon et al. (6), in which
a few Tace
Zn/Zn
pups survived to weaning, we found that
a significant number (25%) of Tace
Zn/Zn
pups survived to
adulthood (8 wk of age) (supplemental Table 2). The ex-
planation for the approximate 2- to 3-fold increase in sur-
vivability of Tace
Zn/Zn
pups is not known but is likely
associated with subtle differences in strain background that
can influence the phenotype of EGFR-deficient mice (29) and
by special attention paid to animal husbandry in the current
study (Materials and Methods).
We previously demonstrated that the adult Tace
Zn/Zn
mice have defective shedding of several well-established
TACE substrates including TNFR1, TNFR2, and l-selectin,
confirming that these mice lack TACE proteolytic activity
(20). As expected, adult Tace
Zn/Zn
mice display many char
-
acteristics previously described in mice with TACE defi-
ciency including open eyelids at birth, stunted and curly
vibrissae, perturbed hair coat, and reduced body weight (6,
12, 14, 30 –32) (supplemental Fig. 1). Specifically, whereas
body weight was normal at birth, both female and male
Tace
Zn/Zn
mice exhibited significantly reduced body weight
by 4 wk of age (Table 1). By 8 wk of age, the magnitude of
this weight difference declined in females but not males.
Body length was also reduced in female and male Tace
Zn/Zn
mice by 19.7 2.5 and 19.3 2.6%, respectively, at 8 wk of
age. By contrast, female and male heterozygous Tace
Zn/
littermates had body lengths and body weights similar
to that of wild-type (WT) controls at all ages examined
(Table 1).
Tace
Zn/Zn
mice have reduced fat mass despite normal
food intake
To determine whether reductions of body fat mass, lean
mass, or both contribute to the reduced body weight of
Tace
Zn/Zn
mice, QMR measurements of body composition
were conducted. At age 12–14 wk, male Tace
Zn/Zn
mice
were characterized by decreases of both total lean and fat
mass compared with controls (Fig. 1, A, C, and D), with the
reduction of fat mass but not lean mass, remaining significant
when normalized to total body weight (30.6% reduction, n
10–14, P 0.05) (Fig. 1, E and F; supplemental Table 3). As
predicted by the reduced fat mass, plasma leptin levels were
TABLE 1. Phenotypic analysis of Tace
Zn/Zn
, Tace
/Zn
, and WT littermates
WT Tace
/Zn
Tace
Zn/Zn
P n
Body length (cm)
8 wk male 9.94 0.11 9.98 0.24 7.98 0.25 0.001 11
8 wk female 9.24 0.10 8.99 0.20 7.46 0.24 0.001 9
Body weight (g)
P1 1.50 0.04 1.56 0.03 1.35 0.09
a
0.05 8
4 wk male 17.38 0.45 18.67 0.48 9.49 0.91 0.001 10
4 wk female 14.26 0.57 14.79 0.38 9.85 1.00 0.001 6
8 wk male 24.44 0.74 24.98 0.78 18.37 1.17 0.001 8
8 wk female 20.40 0.65 21.53 0.76 18.52 0.28
a
0.05 9
a
Tace
Zn/Zn
mice were significantly different only from Tace
/Zn
mice.
Gelling et al. TACE Regulation of Energy Expenditure Endocrinology, December 2008, 149(12):6053–6064 6055
also decreased in Tace
Zn/Zn
mice compared with WT con
-
trols (Fig. 1B). To investigate whether the reduction of
plasma leptin levels and other responses are appropriate
for the decrease of body fat mass, a subset of Tace
Zn/Zn
mice were compared with WT littermates that were ca-
lorically restricted (WT-CR) to achieve a comparable de-
crease of body fat content. As expected, WT-CR mice ex-
hibited reductions of total body weight, lean mass, and fat
mass comparable with that of Tace
Zn/Zn
mice, yet plasma
leptin levels remained lower in TACE-deficient mice than
in the WT-CR group (Fig. 1B). Whereas normal animals
exhibit hyperphagia in response to reduced levels of body
fat mass and plasma leptin (33, 34), food intake of Tace
Zn/Zn
mice was comparable with that of WT mice fed ad libitum
(Fig. 1G).
Reduced WAT content in Tace
Zn/Zn
mice is not due to
defective adipocyte differentiation
TACE proteolytic activity has been linked to the regulation
of several signaling pathways involved in adipocyte differ-
entiation including TNF
and preadipocyte factor 1 (PREF-1)
signaling (35, 36). To determine whether the capacity for
adipocyte differentiation is reduced in Tace
Zn/Zn
mice,
MEFs were isolated from E14.5 wild-type and Tace
Zn/Zn
embryos and subsequently examined for their ability to dif-
ferentiate in vitro. Low-passage, confluent cultures of both
WT control and Tace
Zn/Zn
MEFS exhibited similar patterns
(Fig. 2, A and B) and levels (Fig. 2C) of triglyceride accu-
mulation, as revealed using Oil Red O staining 8 d after
onset of differentiation. In cell culture, therefore, TACE
WT TACE
Zn/
Zn
WT-CR
0
10
20
30
***
#
**
WT TACE
Zn/
Zn
WT-CR
0
5
10
15
20
***
*
WT TACE
Zn/
Zn
WT-CR
0
1
2
3
4
***
*
WT TACE
Zn/
Zn
WT-CR
0
5
10
15
*
A
C
B
D
WT TACE
Zn/
Zn
WT-CR
0
2
4
6
*
WT TACE
Zn/
Zn
WT-CR
0
2
4
6
E
F
FIG.1.Tace
Zn/Zn
mice have reduced fat mass despite normal food intake. QMR measurements were performed on 12- to 14-wk-old male
Tace
Zn/Zn
, WT, and WT-CR mice. A, Body weight (***, WT vs. Tace
Zn/Zn
, P 0.001; **, WT vs. WT-CR; #, Tace
Zn/Zn
vs. WT-CR,
P 0.01). B, Leptin plasma levels (*, WT vs. Tace
Zn/Zn
, P 0.05). C, Fat mass (***, WT vs. Tace
Zn/Zn
, P 0.001; *, WT vs. WT-CR,
P 0.05). D, Lean mass (***, WT vs. Tace
Zn/Zn
, P 0.001; *, WT vs. WT-CR, P 0.05). E, Fat mass as a percentage of total body weight
(*, WT vs. Tace
Zn/Zn
, P 0.05). F, Lean mass as a percentage of total body weight. G, Food intake was measured as described in
experimental procedures. In panels, data are presented as mean
SEM. For A, C, D, E, and F, n 10–14; B, n 4–10; G, n 4 –5.
6056 Endocrinology, December 2008, 149(12):6053– 6064 Gelling et al. TACE Regulation of Energy Expenditure
activity does not appear to be essential for adipocyte
differentiation.
To further investigate adipocyte maturation in Tace
Zn/Zn
mice, histological analysis of WAT was performed. In agree-
ment with QMR fat mass analysis, gross inspection of fat
pads from Tace
Zn/Zn
mice demonstrated a marked reduc
-
tion in size of all WAT depots. H&E sections of gonadal WAT
taken from age-matched female WT and Tace
Zn/Zn
mice
revealed fat cells from TACE-deficient mice that were
smaller in size and had a multilocular appearance with in-
creased eosin staining (Fig. 3, A and B). Consistent with these
observations, the number of fat cells per unit area was in-
creased in Tace
Zn/Zn
mice compared with WT littermates
(Fig. 3C). These phenotypic changes are inconsistent with
failure of adipocyte differentiation and are reminiscent of
those observed in WAT that has been genetically or phar-
macologically altered to increase its metabolic activity (37–
40). To investigate this hypothesis further, we measured
WAT expression of the mitochondrial UCP-1, which is nor-
mally found only in BAT (41, 42). UCP-1 protein was detected
at variable levels in two of five WAT samples taken from
Tace
Zn/Zn
mice but was not detected in any WAT sampled
from control littermates (data not shown). Thus, the appear-
ance of BAT characteristics in WAT from Tace
Zn/Zn
mice is
a finding associated with increased whole-body metabolic
rate in several other mutant mouse models (28, 43, 44).
Tace
Zn/Zn
mice have perturbed energy homeostasis
The finding of reduced fat mass despite normal daily food
intake in Tace
Zn/Zn
mice raises the possibility that energy
expenditure is increased in these animals. To test this hy-
pothesis, indirect calorimetry measurements of whole-ani-
mal energy expenditure were performed based on VO
2
nor
-
FIG. 2. MEFs from Tace
Zn/Zn
mice are not defective in adipocyte
differentiation. Primary MEFs derived from wild-type (A) and
Tace
Zn/Zn
(B) 14.5-d-old embryos were treated with differentiation
mixture (DM) as described under experimental procedures. At d 8
after induction, cells were stained for lipid droplets with Oil Red O.
C, Quantification of neutral lipid (Oil Red O) accumulation during
adipocyte differentiation from wild-type and Tace
Zn/Zn
MEFs
treated at d 8 after induction with or without DM. Data are presented
as mean
SEM from triplicate cultures. Scale,40
m.
FIG. 3. WAT from Tace
Zn/Zn
mice exhibits a multilocular appear
-
ance and increased cellularity. Paraffin-embedded sections of gonadal
WAT from wild-type (A) and Tace
Zn/Zn
(B) littermates stained with
H&E. WAT from Tace
Zn/Zn
mice showed a multilocular appearance
with increased eosin staining. C, Quantitation of the number of adi-
pocytes and nuclei within WAT H&E sections. At least three fields
were evaluated per fat pad. Data are presented as mean
SEM (n
5). Differences between WT and Tace
Zn/Zn
mice were evaluated
with Student’s t test. **, P 0.001. Scale,10
m.
Gelling et al. TACE Regulation of Energy Expenditure Endocrinology, December 2008, 149(12):6053–6064 6057
malized to lean body mass (Fig. 4, A and B). As expected,
WT-CR to achieve fat mass comparable with Tace
Zn/Zn
mice
exhibited a 34 4% reduction in mean dark cycle VO
2
compared with ad libitum-fed WT controls, although this
difference was not observed during the light cycle (when
metabolic rate of both groups is relatively reduced). This
reduction in dark-cycle energy expenditure is a homeostatic
response that conserves energy stores in the face of weight
loss due to energy restriction. By contrast, the energy ex-
penditure of Tace
Zn/Zn
mice was dramatically increased in
both dark (by 1.8 0.1-fold, P 0.001, n 4) and light cycles
(by 2.42 0.1-fold, P 0.001, n 4) compared with WT
controls fed ad libitum (Fig. 4, A and B). A similar outcome
was observed when energy expenditure was expressed ei-
ther as total VO
2
or as VO
2
normalized to total body weight
(supplemental Table 3).
This pattern of markedly increased energy expenditure in
Tace
Zn/Zn
mice is therefore opposite to how normal animals
adapt to reduced body fat stores. In addition, whereas mean
dark cycle respiratory quotient was reduced in WT-CR com-
Dark Light
0
2000
4000
6000
8000
***
###
***
###
**
TACE
Zn/
Zn
WT-CR
WT
Dark Light
0.6
0.8
1.0
*
Dark Light
0
2500
5000
7500
10000
**
**
Dark cycle
27 30 32
0
2000
4000
6000
8000
***
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***
Temperature (
o
C)
Light Cycle
27 30 32
0
2000
4000
6000
8000
***
***
***
Temperature (
o
C)
AB
C
D
EF
500
2500
4500
6500
8500
WT-CR
TACE
Zn/
Zn
WT
Dark cycle
Light cycle
Fed Fed
FIG.4.Tace
Zn/Zn
mice have dramatically increased energy expenditure. A, VO
2
in WT, Tace
Zn/Zn
, and WT-CR mice. Arrows indicate when
WT-CR mice were given access to food as described in experimental procedures. B, Mean VO
2
consumption during dark and light cycles (***,
WT vs. Tace
Zn/Zn
; ###, Tace
Zn/Zn
vs. WT-CR; **, WT vs. WT-CR, P 0.001). C, Mean RER during dark and light cycles (*, WT vs. WT-CR
and Tace
Zn/Zn
vs. WT-CR, P 0.05). D, Mean ambulatory activity during dark and light cycles (**, WT vs. Tace
Zn/Zn
and WT vs. WT-CR,
P 0.01). E, Mean VO
2
consumption at thermoneutrality in the dark cycle (***, WT vs. Tace
Zn/Zn
, P 0.001). F, Mean VO
2
consumption
at thermoneutrality in the light cycle (***, WT vs. Tace
Zn/Zn
, P 0.001). All values are given as mean SEM (n 4 –10/group).
6058 Endocrinology, December 2008, 149(12):6053– 6064 Gelling et al. TACE Regulation of Energy Expenditure
pared with WT controls, consistent with increased use of fat
as an energy source (Fig. 4C), the mean dark and light RER
values in Tace
Zn/Zn
mice were similar to those of WT mice.
Therefore, these animals do not display the adaptive increase
of fat, relative to carbohydrate, oxidation characteristic of
animals in which body fat stores are reduced by energy
restriction. Unlike these metabolic responses, Tace
Zn/Zn
mice exhibited a decrease in nocturnal ambulatory activity
(compared with WT controls fed ad libitum) that resembled
the response of WT-CR mice (Fig. 2D). Thus, increased en-
ergy expenditure in mice lacking TACE occurs despite re-
duced physical activity.
An alternative explanation for the increased energy ex-
penditure of Tace
Zn/Zn
mice is that their hair defect (sup
-
plemental Fig. 1) causes loss of body heat and that a com-
pensatory increase of thermogenesis is required to maintain
core body temperature. To test this possibility, indirect cal-
orimetry measurements of WT control and Tace
Zn/Zn
mice
were performed at temperatures that approach thermoneu-
trality (Fig. 4, E and F). As expected, mean dark and light
cycle oxygen consumption decreased in a temperature-de-
pendent manner in both WT controls and Tace
Zn/Zn
mice,
but mean VO
2
values of Tace
Zn/Zn
mice remained markedly
increased during both dark and light cycles compared with
WT littermate controls at ambient temperatures in which loss
of body heat to the environment is minimal. Therefore, the
hypermetabolic phenotype of Tace
Zn/Zn
mice is not a com
-
pensatory response elicited by excessive loss of core body
heat. Because, in addition, circulating concentrations of thy-
roid hormone were not elevated in these animals (Table 2),
these data collectively exclude increased levels of physical
activity, heat loss, or thyroid hormone in the pathogenesis of
hypermetabolism in TACE-deficient mice.
UCP-1 mRNA and protein levels in BAT from
Tace
Zn/Zn
mice
Prominent among the remaining mechanisms that might
explain increased energy expenditure in Tace
Zn/Zn
mice is
an increase of sympathetic nervous system (SNS) outflow to
thermogenic tissues such as BAT. In this setting, BAT hy-
perplasia, combined with increased expression of UCP-1,
causes energy dissipation as heat and increases whole-body
oxygen consumption. To investigate this hypothesis, BAT
weight and histology were analyzed (Fig. 5, A and B). Al-
though the weight of BAT from Tace
Zn/Zn
mice as a per
-
centage of total body weight did not differ significantly from
controls (WT, 0.93 .05 g vs. WT-CR, 0.94 0.04 vs. Tace
Zn/
Zn
, 1.06 0.05 g, n 5–15), cellularity of BAT from Tace
Zn/
Zn
mice was increased (Fig. 5, A–C). Furthermore, BAT
Ucp-1 mRNA levels were reduced in WT-CR mice, as ex-
pected for animals with depleted body fat reserves, whereas
Ucp-1 mRNA levels in Tace
Zn/Zn
mice did not differ sig
-
nificantly from WT controls (supplemental Fig. 2A). How-
ever, at the protein level, a substantial increase in the ex-
pression of UCP-1 was detected in BAT taken from Tace
Zn/Zn
mice as determined by Western blotting (Fig. 5D), and this was
confirmed by UCP-1 immunostaining in tissue sections from
BAT (supplemental Fig. 2, B–E). This pattern of increased cel-
lularity and UCP-1 expression in BAT from mice lacking TACE
is consistent with a role for increased SNS outflow to BAT in the
lean, hypermetabolic phenotype of these animals.
Effect of TACE deficiency on hypothalamic neuropeptide
gene expression
Because the hypothalamic ARC plays a key role in energy
homeostasis, we sought to determine whether TACE defi-
ciency affects neuropeptide gene expression in this brain
region. We found (using real-time PCR) increased hypotha-
FIG. 5. BAT from Tace
Zn/Zn
mice exhibits increased cellularity and
UCP-1 protein levels. Paraffin-embedded sections of BAT from WT (A)
and Tace
Zn/Zn
(B) littermates stained with H&E. BAT from
Tace
Zn/Zn
mice showed increased eosin staining. C, Calculation of
the number of adipocyte nuclei within BAT H&E sections. At least
three fields were evaluated per fat pad. Data are presented as mean
SEM (n 4–5). Differences between WT and Tace
Zn/Zn
mice were
evaluated with Student’s t test (**, P 0.05). Scale,10
m. D, A
representative example of Western blot analysis for UCP-1 expression
in protein extracts of BAT obtained from Tace
Zn/Zn
and sex-
matched WT control mice.
-Actin was used a protein loading control.
TABLE 2. Neuropeptide mRNA expression profiles in the hypothalamus and circulating levels of centrally regulated hormones
Wild type Tace
Zn/Zn
WT-CR P n
Mch mRNA 100.0 9.7 67.5 10.5 87.8 6.0 NS 5 (5–7)
Trh mRNA 100.0 22.9 154.6 26.6 117.9 23.9 NS 5 (5–7)
Free T
4
(
g/ml)
0.79 0.03 0.50 0.11 1.15 0.29 NS 3 (3–4)
Crh mRNA 100.0 9.0 46.9 8.8 152.7 32.5 0.05
a
0.001
b
5 (5–7)
Corticosterone (ng/ml) 308 35 285 34 701 35 0.001
c
8 (8–10)
a
P values were significantly different between Tace
Zn/Zn
and WT.
b
P values were significantly different between Tace
Zn/Zn
and WT-CR.
c
P values were significantly different between Tace
Zn/Zn
and WT-CR and WT and WT-CR.
Gelling et al. TACE Regulation of Energy Expenditure Endocrinology, December 2008, 149(12):6053–6064 6059
lamic Npy and Agrp and reduced Pomc mRNA levels in both
Tace
Zn/Zn
and WT-CR mice compared with ad libitum-fed
WT controls (Fig. 6). This finding suggests that ARC neurons
sense and respond appropriately to deficient energy stores and
reduced leptin levels in mice with TACE deficiency and there-
fore that defects in other components of the energy homeostasis
system explain the phenotype of Tace
Zn/Zn
mice.
This finding prompted us to measure expression of hy-
pothalamic neuropeptides involved in energy homeostasis
that are expressed outside the ARC as well as circulating
hormones regulated by these peptides (Table 2). Although
melanocyte concentrating hormone (Mch) mRNA levels
tended to be decreased in Tace
Zn/Zn
mice compared with
either WT group, this effect failed to reach statistical signif-
icance. Similarly whereas hypothalamic thyroid-releasing
hormone (Trh) mRNA levels tended to be increased in
Tace
Zn/Zn
mice, this difference also did not achieve statis
-
tical significance. As expected for mice with depleted energy
reserves, both CRH (Crh) mRNA levels and circulating cor-
ticosterone hormone levels were increased in the WT-CR
compared with WT animals. In contrast, Crh mRNA levels
were decreased in Tace
Zn/Zn
mice, despite circulating levels
of corticosterone levels that were similar to those of WT mice
fed ad libitum and were far lower than those of WT-CR mice
(Table 2). Unlike the ARC, therefore, the expression of CRH
mRNA in the hypothalamic paraventricular nucleus of mice
lacking TACE appears to differ from that of WT-CR mice.
To exclude the possibility that alterations in neuronal de-
velopment contributed to these differences in neuropeptide
gene expression and neuroendocrine function, we performed
morphological analysis using Nissl staining of cell bodies
and fibers within the hypothalamus of TACE-deficient mice
and WT controls. This analysis failed to reveal any gross
morphological difference of hypothalamic structure between
WT and Tace
Zn/Zn
mice (supplemental Fig. 3, A and B), nor
did we detect differences with respect to either numbers of
hypothalamic neuropeptide Y (NPY)-positive cell bodies and
fibers within the ARC (supplemental Fig. 3, C and D) or
paraventricular nucleus (supplemental Fig. 3, E–H), or syn-
aptophysin staining in the ARC (supplemental Fig. 3, I and
J). Thus, the dramatic effect of TACE deficiency on energy
homeostasis is unlikely to arise from defective neuronal
development.
Discussion
Through the process of energy homeostasis, changes of fat
mass (induced, for example, by energy restriction) elicit com-
pensatory adjustments of feeding behavior and energy me-
tabolism that favor the return of energy stores to their base-
line level. In the current work, we describe fundamental
defects of energy homeostasis induced by TACE deficiency
that result in a lean, profoundly hypermetabolic phenotype.
The reduced fat mass of Tace
Zn/Zn
mice was accompanied
by decreases of circulating leptin levels and changes of ARC
neuropeptide gene expression (increased Npy and Agrp
mRNA levels and decreased Pomc levels) expected for ani-
mals with depleted body fat stores, yet these mice exhibited
neither the increase of food intake nor the decrease of energy
expenditure that serve to restore depleted fat mass in normal
animals. Instead, Tace
Zn/Zn
mice displayed markedly in
-
creased energy expenditure and consumed food in an
amount that, although comparable with WT controls, was
lower than expected for energy-depleted animals. Together,
these results suggest that TACE deficiency causes a hyper-
metabolic phenotype in which the capacity to transduce key
initial components of the response to depleted fuel stores
(involving leptin and ARC neurons) into appropriate behav-
ioral and metabolic outputs is impaired, resulting in exces-
sive leanness (Table 3).
Previous studies characterizing Tace
Zn/Zn
null mice were
performed on several different strain backgrounds (6, 12, 14,
19, 30, 31, 32, 45, 46, 48). In the original report, Tace
Zn/Zn
mice on a mixed (C57BL/6 129) background exhibited
WT TACE
Zn/
Zn
WT-CR
0.0
2.5
5.0
7.5
**
*
#
WT TACE
Zn/
Zn
WT-CR
0
2
4
6
8
**
*
#
WT TACE
Zn/
Zn
WT-CR
0
1
2
3
4
5
**
**
A
B
C
FIG.6.Tace
Zn/Zn
mice display appropriate neuropeptide expres
-
sion within the ARC of the hypothalamus. Neuropeptide gene ex-
pression in the ARC from WT, Tace
Zn/Zn
and WT-CR littermates
was examined by real-time PCR. A, NPY mRNA levels (**, WT vs.
Tace
Zn/Zn
, P 0.001; *, WT vs. WT-CR; #, Tace
Zn/Zn
vs. WT-CR,
P 0.05). B, Agouti-related peptide (AgRP) mRNA levels (**, WT vs.
Tace
Zn/Zn
, P 0.001; *, WT vs. WT-CR; #, Tace
Zn/Zn
vs. WT-CR,
P 0.05). C, Proopiomelanocortin (POMC) mRNA levels (**, WT vs.
Tace
Zn/Zn
and WT vs. WT-CR, P 0.01). All values are given as
mean
SEM (n 5–7/group).
6060 Endocrinology, December 2008, 149(12):6053– 6064 Gelling et al. TACE Regulation of Energy Expenditure
perinatal lethality due, in part, to defective EGFR receptor
signaling that is likely associated with defects in cardiac
development observed in both HB-EGF- and EGFR-deficient
mice (6, 12, 19, 48). By contrast, mice lacking functional TNF
signaling are viable and fertile indicating that reduced TNF
signaling is unlikely to be the primary factor causing post-
natal lethality observed in Tace
Zn/Zn
mice (21–23). Whether
the defective shedding of other TACE substrates is involved
in the postnatal lethality of Tace
Zn/Zn
mice has not been
determined.
Several studies have demonstrated that some EGFR-defi-
cient mice could survive to weaning (9, 29), as do a subset of
Tace
Zn/Zn
mice, although neither the percentage of surviv
-
ing pups nor whether these animals could survive beyond
weaning was reported (6). Compared with previous studies,
we obtained an estimated 2- to 3-fold increase in the number
of Tace
Zn/Zn
pups surviving to and beyond weaning. In
-
terestingly, the number of surviving Tace
Zn/Zn
null mice
observed in our study resembles that of mice with a com-
pound heterozygous Egfr
wa-2/wa-5
mutation. The latter mice
express hypomorphic (wa-2) and antimorphic (wa-5) Egfr
alleles and, like TACE-deficient mice, have markedly re-
duced EGFR activity (49). Thus, a comparable reduction, but
not the complete absence, of EGFR signaling may explain the
limited survival of both mouse models into adulthood (6).
Whereas differences in strain background can influence the
survival of EGFR-deficient mice (29) (our unpublished ob-
servation), both HB-EGF- and EGFR-deficient mice on con-
genic C57BL/6 backgrounds display a range of survivability,
indicating that other unknown factors influence the survival
of these mice (29, 50) (our unpublished observation). Taken
together, these findings suggest that the increase in the per-
centage of surviving Tace
Zn/Zn
mice in the current study
reflects differences in strain background combined with fac-
tors such as improved husbandry.
One plausible mechanism to explain hypermetabolism of
TACE-deficient mice involves increased heat loss arising
from a combination of reduced body size (which increases
the surface area to mass ratio) (51, 52) and altered hair phe-
notype that in turn triggers a compensatory increase of ther-
mogenesis to maintain core body temperature. Interestingly,
in diacylglycerol acyltransferase 1-deficient mice, which also
display a hair/coat defect and increased energy expenditure,
neither the enhanced oxygen consumption nor the core body
temperature of these mice was altered by changes in ambient
temperature, indicating that the hair defect did not cause
body heat loss (53). Whereas the ambient temperature
needed to achieve thermoneutral conditions for Tace
Zn/Zn
mice has not been defined, we found that indirect calorimetry
measurements performed at temperatures approaching ther-
moneutrality showed a persistently elevated rate of oxygen
consumption in TACE-deficient mice. Combined with our
finding that Tace
Zn/Zn
null mice maintain core body tem
-
perature normally compared with their WT controls (our
unpublished observation), these data suggest the hypermeta-
bolic phenotype of Tace
Zn/Zn
mice is not due to either a
defect in thermoregulation or a compensatory response elic-
ited by loss of core body heat. Similarly, our findings also
exclude hyperthyroidism and increased physical activity as
factors contributing to the hypermetabolic phenotype of
mice lacking TACE.
Several members of the matrix metalloproteinase (MMP)/
ADAM/tissue inhibitor of matrix metalloproteinase (TIMP)
axis are expressed during adipocyte differentiation and are
implicated in this process (36, 54–57). For example, genetic
deficiency or overexpression of ADAM12 can alter adipocyte
differentiation (58 60), and membrane type 1-matrix met-
alloproteinase acts as a dominant adipogenic factor during
WAT development (61). Our current studies, however, sug-
gest that WAT differentiation proceeds normally in the ab-
sence of TACE, because MEFs from Tace
Zn/Zn
mice readily
differentiate into adipocytes in cell culture. Relevant to this
finding are recent studies demonstrating an important role
for TACE in the shedding of PREF-1, in which the cleaved
form of PREF-1 is a negative regulator of adipogenesis (62).
In TACE-deficient mice, the lack of PREF-1 shedding would
be predicted to cause a loss of PREF-1 signaling and therefore
would create permissive conditions for adipogenesis, a result
that would be in agreement with the demonstrated capacity
of Tace
Zn/Zn
MEFs to differentiate into adipocytes. It is
noteworthy that, despite this evidence of normal adipocyte
differentiation ex vivo, WAT taken from adult animals lack-
ing TACE displays features characteristic of BAT. Specifi-
cally, gonadal fat pads from Tace
Zn/Zn
mice were charac
-
terized by increased cellularity and adipocytes with a
multilocular appearance that have variable UCP-1 expres-
sion. Whether alterations in WAT phenotype arise from di-
rect or indirect consequences of TACE deficiency, and the
extent to which the hypermetabolic phenotype of Tace
Zn/Zn
mice arises from increased metabolic activity of WAT, are
important questions for future study.
Developmental defects observed in TACE-deficient mice
have been linked to impaired EGFR and/or TNF
receptor
signaling (6) and raise the possibility that the energy ho-
meostasis phenotype of Tace
Zn/Zn
mice is linked to one or
both of these signaling pathways. Although decreased TNF
shedding is associated with protection against diet-induced
insulin resistance and diabetes (45, 46) and TNF
-deficient
mice have a mild reduction of body weight and fat mass due,
in part, to improved insulin sensitivity (63– 65), these effects
were not associated with changes of food intake or energy
expenditure (63, 64). Interestingly, Tace
Zn/Zn
mice do dis
-
play lower blood glucose levels and improved glucose tol-
TABLE 3. Summary of homeostatic responses to reduced body fat
mass in TACE-deficient mice
Expected response to
reduced leptin and fat
mass
Observed response of
TACE-deficient mice
2 Leptin Yes
2 Ambulatory activity Yes
1 Npy mRNA Yes
ARC responses 1 Agrp mRNA Yes
2 Pomc mRNA Yes
1 Crh mRNA No
1 Corticosterone No
2 UCP-1 in BAT No
1 Food intake No
2 VO
2
No
2 RER No
Gelling et al. TACE Regulation of Energy Expenditure Endocrinology, December 2008, 149(12):6053–6064 6061
erance and are more insulin sensitive (supplemental Table 4
and supplemental Fig. 4), which is consistent with a loss of
TNF
signaling contributing to changes in insulin sensitivity.
However, we believe that the primary cause of increase in-
sulin sensitivity of Tace
Zn/Zn
mice is their reduced body fat
and/or hypermetabolic phenotype. Indeed, at least some
phenotypic features of Tace
Zn/Zn
mice (including perinatal
lethality) are observed in the complete absence of TNFR1 and
TNFR2 receptors (6, 66 –70), suggesting that reduced TNF
/
TNFR signaling alone is unlikely to explain the energy ho-
meostasis phenotype of TACE-deficient mice.
Developmental defects of the heart cause heart enlarge-
ment and impaired cardiac function in both TACE- and
EGFR-deficient mice that could potentially contribute to the
hypermetabolic phenotype in adult Tace
Zn/Zn
mice. How
-
ever, HB-EGF-deficient mice, which display the same cardiac
defects as the Tace
Zn/Zn
mice (12), show only a small re
-
duction in body weight (our unpublished observation). In
addition, Tace
Zn/Zn
mice show a preferential reduction in
total fat mass whereas a distinguishing feature of weight loss
induced by heart failure is a predominant reduction in lean
mass (71). Whereas we cannot rule out the possibility that the
cardiac defects observed in Tace
Zn/Zn
mice contribute to
increased energy expenditure, we believe that they are un-
likely to be its principal underlying cause.
In the developing and adult CNS, protein family receptor
signaling functions in astrocyte development, cell survival
(72), and neuronal precursor migration (73, 74). However, the
complexity of the ErbB ligand/receptor interactions and the
shortened life span of ErbB receptor-deficient mice has thus
far precluded detailed analysis of energy homeostasis in
adult animals. Mice with neuron-specific ablation of ErbB4
(a known TACE substrate) (75) have no overt energy ho-
meostasis defect (76) and the energy homeostasis of pheno-
type of neuron-specific ErbB2-deficient mice has not been
described (77). Transgenic mice ubiquitously overexpressing
TGF
ligand for EGFRs have reduced body weight and fat
mass, but energy expenditure was not perturbed in these
animals (10). Similarly, intracerebroventricular injection of
recombinant TGF
or neuregulin into the third ventricle of
hamsters reversibly inhibited food intake (78). Whereas mice
lacking individual EGFR ligands do not recapitulate the lean,
hypermetabolic phenotype of Tace
Zn/Zn
mice, therefore, the
lack of phenotype in these mice probably reflects the over-
lapping and/or redundant functions of different EGFR li-
gands (10 –13, 79, 80). By contrast, EGFR-deficient mice show
significantly reduced body weights (7–9, 29), and the lean
phenotype of adult Tace
Zn/Zn
mice closely resembles that of
adult compound heterozygous Egfr
wa2/wa5
mice (49) (Thread
-
gill, D., personal communication).
Whereas the above observations raise the possibility that
reduced EGFR signaling contributes to the hypermetabolic
phenotype of mice lacking TACE, EGFR deficiency also has
adverse consequences for brain development that were not
observed in Tace
Zn/Zn
mice. In situ hybridization experi
-
ments have shown Tace mRNA to be widely expressed
throughout the CNS including the hypothalamus, in which
it is expressed in astrocytes, tanycytes, and neurons (81– 84).
Although ErbB receptors are implicated in CNS develop-
ment (7–9, 72–74), morphological analysis reported here
failed to identify differences in fiber or cell body staining
within the adult hypothalamus of Tace
Zn/Zn
mice, nor were
gross architectural changes observed in the brain of these
animals during postnatal development (postnatal d 1, 6, and
14) (Hevner, R., personal communication). Thus, Tace
Zn/Zn
mice do not overtly display the developmental defects seen
in these ErbB-deficient mice, despite the close association
between functional ErbB ligand signaling and TACE activity
in other developmental settings (6, 12–14, 67). The extent to
which defective ErbB signaling within the CNS contributes
to the energy homeostasis phenotype of TACE-deficient mice
is therefore an important question that awaits further study.
In rodents, UCP-1 expression in BAT is dynamically reg-
ulated by changes in metabolic state such as exposure to cold
or changes in food intake. These stimuli differentially control
SNS activity that is responsible for the activation of adren-
ergic receptors and downstream expression of UCP-1 in
brown adipocytes (43, 85). In Tace
Zn/Zn
mice, the increased
cellularity and UCP-1 expression in BAT is consistent with
the possibility that perturbations in SNS outflow to BAT may
contribute to the lean, hypermetabolic phenotype of these
animals. In a similar manner, stimuli such as exposure to cold
or
-adrenergic agonists can also induce WAT to BAT dif-
ferentiation (28, 43). Recently phosphatidylinositol 3-kinase
activation in primary leptin-responsive neurons of the CNS
was shown to increase SNS activity of WAT that lead to WAT
to BAT remodeling, which caused the increased energy ex-
penditure and leanness in these mice (44). Interestingly, the
WAT taken from Tace
Zn/Zn
mice also has a multilocular
appearance with increased cellularity and aberrant UCP-1
expression. Although it is difficult to assign tissue-specific
defects in the regulation of energy homeostasis in global
knockout mice, these findings raise the possibility that
perturbations in SNS outflow to both WAT and BAT may
contribute to the lean, hypermetabolic phenotype of
Tace
Zn/Zn
mice.
The hypermetabolic phenotype of adult TACE-deficient
mice suggests a novel, unexpected and potentially important
role for TACE in energy homeostasis. Indeed, the increased
energy expenditure of Tace
Zn/Zn
mice is among the most
dramatic yet reported in a rodent system. Given the variety
of known TACE substrates, additional insight into mecha-
nisms underlying the effect of TACE deficiency on energy
balance will benefit from studies in which metalloproteinase
activity is selectively inhibited in distinct hypothalamic nu-
clei or specific neuronal subsets. Gene therapy approaches
(such as brain region specific adenoviral gene delivery) to
increase or block TACE activity, and cell type-specific con-
ditional deletion (47) or rescue of TACE activity, should also
help to clarify how TACE activity participates in energy
homeostasis and whether inhibition of TACE signaling has
potential in the future of obesity treatment.
Acknowledgments
We gratefully acknowledge the skilled technical efforts of Sarah
Fitzgerald, Peter Ong-Lim, P. Lenhart, P. Gillispie, S. Hostikka, and M.
Harris and helpful discussions and technical advice from Dr. Ryan
Streeper. Body composition and metabolic studies were performed with
support from the Clinical Nutrition Research Unit at the University of
Washington. We thank Dr. J. Peschon and Dr. R. Black (Amgen) for
6062 Endocrinology, December 2008, 149(12):6053– 6064 Gelling et al. TACE Regulation of Energy Expenditure
providing TACE reagents and for their continued advice and support for
this project.
Received May 23, 2008. Accepted July 30, 2008.
Address all correspondence and requests for reprints to: Dr. Peter
Dempsey, Departments of Pediatrics and Molecular and Integrative
Physiology, University of Michigan, A520 MSRB1, 1150 West Medical
Center Drive, Ann Arbor, Michigan 48109. E-mail: petedemp@
med.umich.edu.
This work was supported by National Institutes of Health Grants
DK59778 and DK63363 (to P.J.D.) and DK52989, NS32273, and DK68384
(to M.W.S.) and the Diabetes Endocrinology Research Center at the
University of Washington (to R.W.G.).
Current address for R.W.G.: Department of Metabolism, KinMed,
Inc., Emery, California 94608.
Current address for A.P.: Peace Health, Eugene, Oregon 97401.
Disclosure Statement: The authors have nothing to disclose.
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endocrine community.
6064 Endocrinology, December 2008, 149(12):6053– 6064 Gelling et al. TACE Regulation of Energy Expenditure
    • "using the EchoMRI tm 3-in-1 Animal Tissue Analyzer (Echo Medical Systems, Houston, TX). Energy Expenditure, Respiratory Quotient, and Ambulatory Activity level were determined continuously over 36 h by indirect calorimetry and food intake continuously monitored using the Comprehensive lab Animal Monitoring System (Columbus Instruments Co., Columbus, OH) also located within the EBGM Core as previously described (Gelling et al. 2008; Morton et al. 2011). To control for the influence of body size variation on total energy expenditure, group comparisons involving this outcome were adjusted for total body mass using analysis of covariance (ANCOVA), as recommended (Kaiyala et al. 2010; Kaiyala and Schwartz 2011). "
    [Show abstract] [Hide abstract] ABSTRACT: The lean body weight phenotype of hepatic lipase (HL)-deficient mice (hl(-/-)) suggests that HL is required for normal weight gain, but the underlying mechanisms are unknown. HL plays a unique role in lipoprotein metabolism performing bridging as well as catalytic functions, either of which could participate in energy homeostasis. To determine if both the catalytic and bridging functions or the catalytic function alone are required for the effect of HL on body weight, we studied (hl(-/-)) mice that transgenically express physiologic levels of human (h)HL (with catalytic and bridging functions) or a catalytically-inactive (ci)HL variant (with bridging function only) in which the catalytic Serine 145 was mutated to Alanine. As expected, HL activity in postheparin plasma was restored to physiologic levels only in hHL-transgenic mice (hl(-/-)hHL). During high-fat diet feeding, hHL-transgenic mice exhibited increased body weight gain and body adiposity relative to hl(-/-)ciHL mice. A similar, albeit less robust effect was observed in female hHL-transgenic relative to hl(-/-)ciHL mice. To delineate the basis for this effect, we determined cumulative food intake and measured energy expenditure using calorimetry. Interestingly, in both genders, food intake was 5-10% higher in hl(-/-)hHL mice relative to hl(-/-)ciHL controls. Similarly, energy expenditure was ~10% lower in HL-transgenic mice after adjusting for differences in total body weight. Our results demonstrate that (1) the catalytic function of HL is required to rescue the lean body weight phenotype of hl(-/-) mice; (2) this effect involves complementary changes in both sides of the energy balance equation; and (3) the bridging function alone is insufficient to rescue the lean phenotype of hl(-/-)ciHL mice. © 2015 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and the Physiological Society.
    Full-text · Article · Apr 2015
    • "miR 106b-93 was considered a negative regulator of brown adipocyte differentiation-while miR17-92 were found to promote white adipocyte differentiation of 3TL1 preadipocytes, Wu et al. described a negative effect of miR106b and miR 93 on brown adipogenesis [40]. While miR26a/b induced and promoted human brite adipocyte differentiation, mechanistically the combination of transcriptomics, an RNAi screen and reporter assays revealed that the effects of miR26a/b are largely mediated via its target ADAM metallo peptidase17 (ADAM17/TACE), a gene that was previously described to negatively regulate nonshivering thermogenesis [41,42]. "
    [Show abstract] [Hide abstract] ABSTRACT: MicroRNA’s are small ,approximately 22nuclwotide RNA molecules that post transcriptionally regulate protein coding gene expression by binding to their fully or partially complementary sequences with in target messenger RNA( mRNA)Over the past decade it has become clear that that these noncoding RNA’s function as important regulators of a wide range of cellular processesby modulating gene expression.In animals ,most investigated miRNA’s form imperfect hybrids with sequences in the 3’untranslated region( 3’UTR)with the miRNA 5’-proximal seed region providing most of the pairing specificity.Todate several miRNA;s have been shown to regulate lipid metabolism,including miR122,miR370.miR378/378*,miR758,miR106,miR33.Further recently it has been postulated that circulating miRNA’s could act as biomarkers and may be useful for detection of disease process even before initiation eg disease of adipose tissue,heart,liver and also can act as markers for various diseases like tumours,heart diseae,tissue damage,diabetes mellitus,obesity,acute sepsis .Currently miravensen is one of the modified LNA that has been clinically tried in cases of hepatitis C infection and but for a little doubt of renal toxicity it has faired well in phase 3 trials.Further it can act as a marker for HCV induced Hepatocellular carcinoma.This knowledge is further being used to prevent cardiovascular diseases by increasing HDL by targeting miR33 and miR144.Further dietary modifications on basis of understanding of metabolism is being used to help prevent various diseasesand understanding cholesterol metabolism and role of miRNA’S will help preventing various neurodegenerative diseases and kidney disease pathology besides early detection of breast cancer .Similarly the toxicity associated with statin use can be prevented by simultaneous use of antimiR33 oligonucleotides.Still there are hurdles like tissues where these oligonucleotides don’t penetrate well like adipose tissue and multiple miRNA being targeted and several challenges as highlighted remain to be solved.
    Full-text · Article · Mar 2015
    • "miR 106b-93 was considered a negative regulator of brown adipocyte differentiation-while miR17-92 were found to promote white adipocyte differentiation of 3TL1 preadipocytes, Wu et al. described a negative effect of miR106b and miR 93 on brown adipogenesis [40]. While miR26a/b induced and promoted human brite adipocyte differentiation, mechanistically the combination of transcriptomics, an RNAi screen and reporter assays revealed that the effects of miR26a/b are largely mediated via its target ADAM metallo peptidase17 (ADAM17/TACE), a gene that was previously described to negatively regulate nonshivering thermogenesis [41,42]. "
    Full-text · Article · Jan 2015 · Health & medicine: journal of the Health and Medicine Policy Research Group
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