![aP2-MSTN transgenic mice have an increased metabolic rate with increased glycolysis in their adipose tissue compared with WT littermates. ( A ) Transgenic (Tg) and WT littermates were placed in metabolic cages that simultaneously measures metabolic rate (as calculated by gas exchange), activity, food intake, and weight. Transgenic animals had an increased metabolic rate ( P ϭ 0.005), slightly increased food intake, and similar activity as WT littermates. This increased metabolic rate likely contributes to protecting the animals from obesity. ( B ) Quantitative real-time PCR was used to measure the expression levels of genes involved in glucose utilization in the adipose tissue of the mice. Transgenic animals had higher expression levels than WT littermates of genes involved in glucose transport (GLUT1 and GLUT4) and enzymes in the glycolysis pathway [glucokinase (GK), hexokinase (HK), and pyruvate kinase (PK)]. In addition, the aP2-MSTN animals had lower expression levels of some of the genes involved in lipogenesis [fatty acid synthase (FAS) and diacylglycerol acyltransferase 2 (DGAT2)]. ( C ) The rate of glucose oxidation in adipocytes differentiated in tissue culture was directly measured by using 14 C glucose (see Methods ). Adipocytes generated after exposure to MIM have a cell autonomous higher rate of glucose oxidation than adipocytes generated after DIM exposure ( P ϭ 0.001). Error bars represent SDs.](https://www.researchgate.net/profile/Keith-Yamamoto/publication/6764038/figure/fig4/AS:281511522783236@1444128985905/aP2-MSTN-transgenic-mice-have-an-increased-metabolic-rate-with-increased-glycolysis-in_Q320.jpg)
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Myostatin modulates adipogenesis to generate
adipocytes with favorable metabolic effects
Brian J. Feldman*
†
, Ryan S. Streeper
‡
, Robert V. Farese, Jr.
द
, and Keith R. Yamamoto
†储
Departments of *Pediatrics,
†
Cellular and Molecular Pharmacology,
§
Medicine, and
¶
Biochemistry and Biophysics, University of California,
San Francisco, CA 94143; and
‡
Gladstone Institute of Cardiovascular Disease, San Francisco, CA 94143
Contributed by Keith R. Yamamoto, August 28, 2006
A pluripotent cell line, C3H10T1兾2, is induced to undergo adipo-
genesis by a mixture of factors that includes a glucocorticoid such
as dexamethasone. We found that expression of myostatin
(MSTN), a TGF-

family member extensively studied in muscle, was
induced by dexamethasone under those differentiation conditions.
Moreover, MSTN could substitute for dexamethasone in the adi-
pogenesis mixture. However, the adipocytes induced by MSTN in
both cell culture and transgenic mice were small and expressed
markers characteristic of immature adipocytes. These adipocytes
exhibited cell-autonomous increases in insulin sensitivity and glu-
cose oxidation. In mice, these effects produced elevated systemic
insulin sensitivity and resistance to diet-induced obesity. Modula-
tion of the final stages of adipogenesis may provide a novel
approach to understanding and treating metabolic disease.
diabetes 兩 obesity
O
besity is associated with a metabolic imbalance that pro-
duces increased adipose tissue mass caused by a combina-
tion of hypertrophy and hyperplasia of adipoc ytes (1). Impor-
t antly, adipose tissue is itself a critical regulator of systemic
met abolism (2), which suggests that modulation of adipogenesis
should have a greater systemic impact than merely altering
adipose tissue mass. Indeed, underst anding the signals involved
in regulating adipogenesis might have broad implications for
diseases such as diabetes.
Adipoc ytes originate from pluripotent progen itor cells that
are recruited to the adipocyte fate (3, 4). A group of factors and
c ytokines, not fully defined, initiate a differentiation cascade in
which a stem cell commits to a lineage, for example, to become
a ‘‘preadipocyte.’’ Further signals continue the progression by
inducing proliferation and finally differentiation into lipid-laden
adipoc ytes (5). To better define these signals, tissue culture
models of adipogenesis have been generated. These studies have
identified a ‘‘mixture’’ of factors that includes dexamethasone,
insulin, and isobut ylmethylxanthine (DIM) that can trigger
adipogenesis in cell culture (5). However, specific roles for each
c omponent of this mixture are poorly understood.
Myost atin (MSTN), also known as growth and differentiation
factor 8 (GDF8), is a member of the TGF-

super family. MSTN
function has been extensively studied in muscle tissue (6, 7),
whereas its role in adipose tissue is not well understood. For
example, dexamethasone induces MSTN expression in muscle
cells (8), but it was not known whether this regulation also occurs
during adipogenesis, or whether MSTN contributes to the dif-
ferentiation process. Consistent with a role for MSTN in early
st ages of adipogenesis, reduction of MSTN expression or activit y
results in decreased adipose tissue in mice (9–11). In addition,
other studies suggested that MSTN plays a role in cell fate
decisions, biasing differentiation toward the adipocyte lineage
and away from the muscle lineage (12). Paradoxically, however,
studies of MSTN expression and action in preadipocytes imply
that MSTN inhibits rather than promotes the formation of
mature adipocy tes (13, 14).
We sought to probe the relationship between MSTN and the
dexamethasone component of the DIM mixture and better
underst and the functions and actions of these signals. We studied
how these signals modulate adipogenesis in cell culture and mice
and evaluated the cellular and systemic consequences of their
actions.
Results
MSTN Is Induced by Dexamethasone in C3H10T1兾2 Cells and Contrib-
utes to Adipogenesis.
We first examined whether MSTN is in-
duced by dexamethasone in the C3H10T1兾2 cell line, which can
dif ferentiate into adipose, muscle, bone, or cartilage under
specific conditions (15); thus, C3H10T1 兾2 displays characteris-
tics of mesenchymal stem cells. DIM induces adipogenesis in this
cell line (16). The synthetic glucocorticoid dexamethasone is an
essential component of the mixture, as the c ombination of
insulin and isobutylmethylxanthine did not lead to adipogenesis
(Fig. 1). By quantitative real-time PCR, we found that MSTN
ex pression was undetectable in C3H10T1兾2 in the absence of
dexamethasone, and that it was significantly induced (⬎100-fold)
af ter a 4-h exposure to dexamethasone. Thus, MSTN expression
is strongly regulated in C3H10T1兾2 cells by the gluc ocorticoid
c omponent of the differentiation mixture.
We then examined whether adding rec ombinant purified
MSTN could substitute for dexamethasone in the DIM mixture
(MIM). As shown in Fig. 1, the MIM mixture induced significant
levels of adipogenesis as defined by the ac cumulation of intra-
cellular lipid droplets as assessed by Oil-Red-O staining (13).
For comparison, we examined 3T3-L1 cells, a widely studied
cell line that is further differentiated than the C3H10T1兾2 cells
because it is committed to the adipocyte lineage. Although
3T3-L1 has not completed differentiation and therefore is
c ommonly called a preadipocyte cell line, it can be ef ficiently
dif ferentiated to mature adipocytes by the DIM mixture. Unlike
our finding in C3H10T1兾2 cells, the DIM mixture but not the
MIM mixture induced adipogenesis in 3T3-L1 cells (Fig. 1). This
result is consistent with a prior report studying MSTN action in
this cell line (13). Taken at face value, our results suggest that
MSTN can induce adipogenesis as part of the MIM mixture if it
is presented to very-early-stage cells such as mesenchymal stem
cells, but that it has lost its efficacy in adipocyte-c ommitted cells
such as 3T3-L1.
MSTN-Induced Adipocytes Are Small and Apparently Immature. In-
terestingly, the C3H10T1兾2 adipocytes induced by MIM were
smaller than those produced by DIM treatment (Fig. 2). To study
these small adipoc ytes further, we measured the expression of a
series of adipocyte-specific genes, three of which [peroxisome
Author contributions: B.J.F. and K.R.Y. designed research; B.J.F. and R.S.S. performed
research; R.V.F. contributed new reagents兾analytic tools; B.J.F. and K.R.Y. analyzed data;
and B.J.F. and K.R.Y. wrote the paper.
The authors declare no conflict of interest.
Freely available online through the PNAS open access option.
Abbreviations: MSTN, myostatin; DIM, dexamethasone兾insulin兾isobutylmethylxanthine;
MIM, MSTN兾insulin兾isobutylmethylxanthine; aP2, adipocyte P2.
储
To whom correspondence should be addressed. E-mail: yamamoto@cmp.ucsf.edu.
© 2006 by The National Academy of Sciences of the USA
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proliferator-activator receptor
␥
, the fatty acid-binding protein
adipoc yte P2 (aP2), and lipoprotein lipase] exhibit elevated
ex pression levels in mature adipocytes, whereas one (preadipo-
c yte factor 1) is expressed at lower levels in mature adipocytes
c ompared with earlier stages in adipogenesis (16, 17). The small
adipoc ytes induced by MSTN expressed these markers in a
pattern similar to that observed before completion of adipogen-
esis (Fig. 3A), suggesting that they may be immature adipoc ytes.
Mature adipose tissue displays endocrine functions, secreting
a number of adipokines (2). Therefore, we measured adipokine
ex pression in the adipoc ytes generated after MSTN exposure,
c ompared with that in adipoc ytes induced by dexamethasone.
The small adipocytes expressed lower levels of adipok ines than
those induced by dexamethasone, further supporting the hypoth-
esis that the MSTN-induced adipocytes are immature (Fig. 3A).
Adipocytes in aP2-MSTN Transgenic Mice. We next investigated the
in vivo relevance of our in vitro findings by generating transgenic
mice that express MSTN in cells undergoing adipogenesis;
specifically, we constructed a MSTN transgene under the c ontrol
of the aP2 promoter and regulatory element (aP2-MSTN). This
promoter is active in the C3H10T1兾2 mesenchymal cell line and
mouse bone marrow and remains active throughout adipogenesis
(Fig. 4A). Use of this promoter and regulatory element has also
been validated in other animal models; for example, aP2-Wnt10b
mice, generated with the same transgene promoter and enhancer
element, display altered mesenchymal stem cell fate (18).
A lthough aP2-MSTN mice were smaller than their WT litter-
mates (Fig. 4C), they otherwise appeared proportional, healthy,
and without gross malformations. However, adipose tissue f rom
the aP2-MSTN transgenic mice displayed a gene expression
profile and reduced cell size similar to the adipocytes differen-
tiated by the MIM mixture in vitro (Fig. 3A and Fig. 6, which is
published as supporting information on the PNAS web site). This
profile was in contrast to the expression pattern found in the
adipose tissue from the WT mice, which, as expected, displayed
the mature adipocyte profile. Consistent with these findings, we
showed that serum levels of the adipokine, adiponectin, were
reduced in the aP2-MSTN mice relative to WT (Fig. 3B).
Therefore, both in vitro and in vivo, MSTN leads to the formation
of adipocy tes with a distinct ex pression profile.
Metabolic Effects of MSTN Transgene Expression in Adipose Tissue.
We next investigated the systemic metabolic c onsequences of the
altered adipose tissue in aP2-MSTN mice. We performed glu-
c ose tolerance tests on lean mice fed regular chow. Compared
with their WT littermates, the transgenic animals exhibited
sign ificantly higher levels of insulin sensitivity, as demonstrated
by their lower fasting glucose levels, lower hyperglycemia, and
more rapid return to euglycemia (Fig. 4B Upper).
We next performed gluc ose tolerance tests on mice fed a
high-fat diet for 7 weeks. WT animals showed evidence of insulin
resistance as demonstrated by hyperglycemia and delayed return
to euglycemia after receiving a glucose bolus. This finding is
c ommon in mice and humans with insulin resist ance and is
c ommonly used as a diagnostic test for type II diabetes in humans
(19). In striking contrast, the aP2-MSTN transgenic mice main-
t ained normal insulin sensitivity on the high-fat diet, as assessed
by their lower fasting blood glucose and normal glucose toler-
ance tests (Fig. 4B Lower).
Adipocyte Autonomous Increase in Insulin Sensitivity with MSTN
Transgene Expression. To determine whether systemic insulin
sensitivity could be attributed to the altered adipocytes, primary
adipoc ytes were purified from WT and transgenic animals, and
insulin sensitivity was tested by measuring upt ake of [
3
H]2-
deoxyglucose after exposure to insulin. Adipocytes harvested
f rom WT an imals responded to insulin with a 3-fold increase in
intracellular gluc ose concentration, whereas adipocytes f rom the
aP2-MSTN animals displayed a 7-fold increase. These results
suggest that the insulin sensitivity of the aP2-MSTN mice is, at
least in part, a cell-autonomous ef fect in adipocytes. To assess
the possibility that a systemic factor (such as obesity) was
af fecting these results, the same experiment was perfor med on
C3H10T1 兾2 cells that had been differentiated into adipocy tes in
vitro with either MIM or DIM. Adipocytes generated in culture
with MIM were 3-fold more insulin sensitive than DIM-induced
adipoc ytes.
Favorable Systemic Insulin, Glucose, and Triglyceride Levels in aP2-
MSTN Mice. To test whether the immature adipocytes were
protective against other metabolic derangements, we measured
fasting insulin, gluc ose, and triglyceride levels in the aP2-MSTN
mice compared with age-matched WT animals, both maint ained
Fig. 1. MIM induced adipogenesis in C3H10T1兾2 cells but not in 3T3-L1 cells.
Either C3H10T1兾2(Upper) or 3T3-L1 (Lower) cells were grown to confluence.
Cells were then exposed to isobutylmethylxanthine and insulin alone (IM,
Left) or with DIM (Center) or MIM (Right). After 6–10 days of culture, cells were
fixed and stained with Oil-Red-O to identify adipocytes. IM was not suffi-
cient to induce adipogenesis in either cell line. DIM induced adipogenesis
in both cell lines. In C3H10T1兾2 cells, but not in 3T3-L1 cells, MIM induced
adipogenesis.
Fig. 2. MSTN induced adipogenesis in C3H10T1兾2 cells. Adipogenesis was
induced in confluent C3H10T1兾2 cells with either MIM (A, C, and E) or DIM (B,
D, and F). Cells were photographed under light microscopy while in culture (A
and B) or after fixation and staining with Oil-Red-O (C–F). Adipocytes formed
after MSTN treatment were smaller and appeared to contain less lipid than the
adipocytes formed after dexamethasone treatment. Cells exposed to either
isobutylmethylxanthine and insulin or each component alone had minimal
adipogenesis (data not shown). (Magnification: A, B, E, and F, ⫻200; C and D,
⫻100.)
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on a high-fat diet. The transgenic animals did not display any of
the characteristics of metabolic syndrome that were evident in
the WT mice. In particular, the transgenic mice had significantly
lower fasting insulin, glucose, and triglyceride levels than WT
mice (Fig. 4D).
aP2-MSTN Transgenic Mice Have an Increased Metabolic Rate and Are
Resistant to Diet-Induced Obesity. Energy balance was examined in
lean (chow-fed) transgenic and WT littermate mice in metabolic
cages, which continuously measure food intake, locomotor ac-
tivity, and metabolic rate as calculated by oxygen consumption
(VO
2
). aP2-MSTN mice had a higher metabolic rate than WT
mice (P ⫽ 0.005), whereas activit y and food intake (normalized
to total body mass) were similar (Fig. 5A). This finding suggested
that the transgenic animals might be resistant to diet-induced
obesity. Consequently, transgenic and c ontrol animals were
placed on the high-fat diet for 7 weeks, and the quantity of
adipose tissue was monitored by dual energy x-ray absorptiom-
etry scanning. As ex pected, WT mice became obese on this diet
with a mean body fat of 38%, whereas the aP2-MSTN mice were
resistant to weight gain, reaching a maximum mean body fat of
26% (Fig. 4C). Therefore, the adipocytes present in the aP2-
MSTN animals appear to produce a higher met abolic rate and
resistance to the development of obesity.
MSTN Transgene Expression Leads to Increased Glucose Oxidation.
We next studied oxygen consumption and aerobic metabolism in
lean animals, using met abolic cages to measure the respiratory
exchange ratio (VCO
2
兾VO
2
). Although not statistically signifi-
cant, the ratio was greater in transgenic animals than in WT
an imals (Fig. 5A), which suggested that the aP2-MSTN mice may
oxidize glucose to generate energy to a greater extent than the
WT mice. Consistent with this view, quantitative real-time PCR
revealed increased ex pression of mRNA-enc oding glucose trans-
porters GLUT1 and GLUT4, and critical glycoly tic enzymes
hexok inase, glucokinase, pyruvate k inase, and pyr uvate dehy-
drogenase, in adipose tissue from aP2-MSTN mice. Further-
more, mRNAs for fat biosynthesis enzy mes such as fatty acid
synthase and acyl CoA:diacylglycerol acyltransferase 2 were
down-regulated in the transgenic adipose tissue compared with
WT (Fig. 5B). These results suggest that the increased metabolic
rate and resistance to obesity found in the transgenic an imals
reflects increased glucose uptake and glucose oxidation in the
adipose tissue.
To determine whether MSTN-induced adipocytes use more
gluc ose in a cell autonomous manner, we directly measured
gluc ose oxidation, using
14
C-labeled glucose (see Methods), in
C3H10T1 兾2 cells that had been differentiated to adipocytes with
MIM exposure compared with adipoc ytes generated by DIM
treatment. As predicted, the adipocytes generated after MSTN
treatment had sign ificantly (P ⫽ 0.001) more glucose oxidation
than dexamethasone-induced adipocytes (Fig. 5C).
Discussion
We have demonstrated that MSTN is induced by dexamethasone
in mesenchymal stem cells, and that it can substitute for dexa-
methasone to induce adipogenesis. In c ontrast, MSTN fails to
trigger adipogenesis in the 3T3-L1 preadipocyte cell line. We
speculate that a sensitive interval may exist during cell fate
deter mination and early differentiation of mesenchymal stem
cells in which MSTN can induce adipogenesis, and that the
sensitive period ends before the preadipocyte stage represented
Fig. 3. Adipocytes generated with MSTN exposure have the expression profile of an immature adipocyte. (A) Real-time quantitative PCR was used to compare
the expression patterns between adipocytes formed in culture after MIM or DIM exposure (n ⫽ 4 for each condition). (Upper Left) MSTN-induced adipocytes had
lower levels of mature adipocyte markers peroxisome proliferator-activator receptor
␥
(PPAR), aP2, and lipoprotein lipase (LPL) and a higher level of the immature
marker preadipocyte factor 1 (Pref-1). (Upper Right) Cultured MSTN-induced adipocytes also had lower expression levels of adipokines compared with
dexamethasone-induced adipocytes. (Lower) MSTN transgenic (TG) mice had an analogues immature adipose tissue expression profile and lower levels of
adipokine expression. (B) Transgenic animals had lower levels of circulating adiponectin as measured by ELISA performed on serum from transgenic (Tg) mice
and WT littermates. Error bars represent SDs.
Feldman et al. PNAS
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PHYSIOLOGY
by the 3T3-L1 cells. This hypothesis could explain the seemingly
c onflicting effects of MSTN reported previously. In addition,
this hypothesis is c onsistent with the findings in the MSTN
k nockout mice. These animals have decreased adipose tissue,
which may result from impaired initiation of adipogenesis.
Import antly, adipogenesis induced in C3H10T1兾2 by MSTN
yielded cells that maint ain the ex pression profile of immature
adipoc ytes. Furthermore, expression of MSTN during adipogen-
esis in vivo produced similar results, with the adipose tissue in
these mice maintaining this distinct expression pattern. We
postulate that the adipoc ytes generated after MSTN exposure
represent a novel stage in adipogenesis that is further differen-
tiated than the preadipoc yte but earlier than the fully differen-
tiated adipocyte. According to this model, dexamethasone-
c ontaining dif ferentiation conditions such as the DIM mixture
would trigger cells to pass through this stage, whereas MSTN-
c ontaining conditions such as the MIM mixture would lead to
ac cumulation of the immature adipocytes. Our findings in the
MSTN transgen ic mice suggest that MSTN expression may
dominantly inhibit this later stage in adipogenesis, as the an imals
are, of course, glucocorticoid replete. Indeed, we have observed
such dominant negative behavior of MSTN in C3H10T1兾2 cells
(B.J.F., unpublished results). In future studies, it will be inter-
esting to examine the mechanistic relationships of the glucoc or-
tic oid and MSTN signals in adipogenesis and determine whether
the MSTN-induced cells are precursors along the full differen-
tiation pathway, or rather represent a branch that exits from the
pathway.
Finally, it is notable that the MSTN-induced adipocytes have
a favorable impact on metabolism. In particular, metabolic
studies revealed that these changes result in improved insulin
sensitivity and resistance to obesity, which results, at least in part,
f rom increased glucose oxidation. These findings suggest that
altering the final st ages of adipogenesis, for example, through
Fig. 4. aP2-MSTN mice have a favorable metabolic profile and are resistant to obesity. (A) The transgene (Tg) construct was created by subcloning the full-length
MSTN cDNA behind an aP2 promoter. Transgenic mice had a 10-fold overexpression of MSTN as detected by quantitative real-time PCR amplification of cDNA
generated from the adipose tissue of WT and transgenic mice. Primers P1 and P2 were used to amplify a transgene-specific cDNA, whereas primers P1 and P3
were used to compare total MSTN expression levels. Quantitative PCR amplification of hypoxanthine-guanine phosphoribosyl transferase (HPRT) was used to
control for cDNA concentration between samples. (B) Glucose tolerance tests were performed on both lean mice (Upper) and mice on a high-fat diet (Lower).
Blood sugar levels were checked after an overnight fast (time ⫽ 0) and then 2 g兾kg of glucose was injected i.p. After the glucose bolus, tail blood sugar levels
were checked at each time point. At the 60-min time point, all WT animals on the high-fat diet had blood sugars above the upper limit of the meter, therefore
the upper limit value (600 mg兾dl) was used at that time point. aP2-MSTN mice were more insulin sensitive than their WT littermates on both regular (P ⫽ 0.0061)
and high-fat chow (P ⫽ 0.0025). (C) aP2-MSTN mice and their WT littermates were placed on a high-fat diet for 7 weeks. Transgenic mice (Lower Right) were
resistant to the obesity that developed in WT animals (Lower Left). (D) aP2-MSTN mice had lower fasting glucose, insulin, and triglyceride levels than their WT
littermates. Error bars represent SDs.
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t argeted MSTN ex posure, may have a therapeutic benefit for
humans with metabolic diseases such as diabetes.
Methods
Tissue Culture and Differentiation Assays. C3H10T1兾2 cells were
obt ained from the Un iversity of California, San Francisco tissue
culture core facility. 3T3-L1 cells were obtained from ATCC
(Manassas, VA). Cells were grown to confluence in high-gluc ose
DMEM supplemented with 1 mM pyr uvate and 10% FBS. Two
days af ter c onfluence cells were exposed to either 1
M dexa-
methasone (Sigma, St. Louis, MO) or 40 nM recombinant MSTN
(R&D Systems, Minneapolis, MN) in addition to 11.5
g兾ml
isobut ylmethylxanthine (Sigma) and 24 ng兾ml insulin (Sigma).
Cells were cultured for 10 days, and half of the media was
replaced with fresh DMEM and 10% FBS every 2 days.
Quantitative PCR. Quantitative PCR was perfor med as described
(20) with an Opticon2 machine and sof tware (MJ Research,
Cambridge, MA). Primer sequences used are available on re-
quest.
Generation of Transgenic Mice. MSTN cDNA was amplified from
mouse muscle RNA (Stratagene, La Jolla, CA). The cDNA was
subcloned behind the aP2 promoter and regulatory domain and
in front of a poly(A) sequence (gifts from Ormond MacDougald,
Un iversity of Michigan, A nn Arbor, MI). Transgene microin-
jection and transplant into pseudopregnant mice was performed
by the Un iversity of California, San Francisco transgenic cancer
c ore facility using the FVB strain. Genot yping was done by PCR
using transgene-specific primers. A ll mice were housed in a
pathogen-f ree barrier-type facility (12-h light兾12-h dark cycle).
Male mice were used for all studies, and they were fed either a
st andard chow diet (5053 PicoLab Diet; Purina, St. Louis, MO)
or a high-fat, Western-type diet (TD.01064, Harlan-Teklad,
Madison, WI) that c ontains 20% anhydrous milk fat, 1% corn
oil, and 0.2% cholesterol by weight. All an imal studies were
approved by the Un iversity of Californ ia, San Francisco Com-
mittee on Animal Research.
Glucose Tolerance Testing. Male transgenic (n ⫽ 5) and WT (n ⫽
7) littermate animals, at 3–4 months of age, were fasted over-
n ight. Fasting and subsequent glucose levels were obtained from
t ail vein blood with a One Touch Ultra glucometer and test
strips. Glucose (2 g兾kg) dissolved in sterile saline was injected
into the peritoneal cavit y of the mice, and blood glucose was
mon itored.
Isolation of Primary Adipocytes and Determination of Glucose Trans-
port. Insulin-stimulated glucose transport in purified primary
white adipocytes from transgenic and WT littermates was de-
ter mined as described (1, 21). Briefly, fat depots where treated
with t ype I collagenase (2 mg兾ml; Worthington Biochemical,
L akewood, NJ) and filtered through 500-
m nylon mesh. Adi-
poc ytes were washed three times with Krebs-Ringer-Hepes
buf fer and 2.5% BSA. Cells were incubated with [
3
H]2-
deoxyglucose with and without insulin for 30 min. Adipocytes
Fig. 5. aP2-MSTN transgenic mice have an increased metabolic rate with increased glycolysis in their adipose tissue compared with WT littermates. (A)
Transgenic (Tg) and WT littermates were placed in metabolic cages that simultaneously measures metabolic rate (as calculated by gas exchange), activity, food
intake, and weight. Transgenic animals had an increased metabolic rate (P ⫽ 0.005), slightly increased food intake, and similar activity as WT littermates. This
increased metabolic rate likely contributes to protecting the animals from obesity. (B) Quantitative real-time PCR was used to measure the expression levels of
genes involved in glucose utilization in the adipose tissue of the mice. Transgenic animals had higher expression levels than WT littermates of genes involved
in glucose transport (GLUT1 and GLUT4) and enzymes in the glycolysis pathway [glucokinase (GK), hexokinase (HK), and pyruvate kinase (PK)]. In addition, the
aP2-MSTN animals had lower expression levels of some of the genes involved in lipogenesis [fatty acid synthase (FAS) and diacylglycerol acyltransferase 2
(DGAT2)]. (C) The rate of glucose oxidation in adipocytes differentiated in tissue culture was directly measured by using
14
C glucose (see Methods). Adipocytes
generated after exposure to MIM have a cell autonomous higher rate of glucose oxidation than adipocytes generated after DIM exposure (P ⫽ 0.001). Error bars
represent SDs.
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were purified from media by passing through silic on oil DC550
with phthalic acid dinonyl ester (ratio 2:3). Intracellular [
3
H]2-
deoxyglucose was quantified with a scintillation counter. Adi-
poc yte yield was variable between samples, therefore, insulin
sensitivity was internally normalized by calculating fold change
bet ween basal and insulin-induced samples.
Rate of Glucose Oxidation. Rate of glucose oxidation was deter-
mined as described (22). Briefly, confluent C3H10T1兾 2 cells
were treated with MIM, DIM, or vehicle and cultured for 10 days
as described. Cells were washed and st arved for2hinKrebes-
Ringer-Hepes buffer. C
14
gluc ose alone (basal) or with insulin
was added to the media and cells were incubated at 37°C for 2 h.
14
CO
2
was released with the addition of 2 M HCL and quantified
by scintillation counting.
Body Composition. Mice were fasted for 4 h and anesthetized with
isoflurane, and their body composition was analyzed by dual
energy x-ray absorptiometry with a PixiMus2 scanner (GE
Healthcare Lunar, Madison, WI).
Energy Balance. Food intake and oxygen consumption (VO
2
)
were measured by indirect calorimetry (Oxymax Comprehensive
L ab An imal Monitoring System, Columbus Instruments, Co-
lumbus, OH) over 3 days. Both parameters were normalized to
lean body mass, as measured by dual energy x-ray absorptiom-
etry scanning on the day of initiating calorimetry studies. Studies
were performed on three WT and five transgen ic male mice. P
values were calculated by using two-tailed t tests.
We thank Ormand MacDougald for providing the aP2 promoter and
enhancer construct; Wally Wang, Stefan Taubert, and David Feldman
for comments on the manuscript; members of K.R.Y.’s laboratory for
valuable discussions; and Valerie Doughert y for administrative support.
This work was supported by National Institutes of Health Grants
CA20535 (to K.R.Y.), DK56084 (to R.V.F.), DK07161 and DK73697 (to
B.J.F.), and DK56084 (to R.S.S.).
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