Article

Targeted Loss of GHR Signaling in Mouse Skeletal Muscle Protects Against High-Fat Diet–Induced Metabolic Deterioration

Division of Endocrinology, Diabetes, and Bone Diseases, Department of Medicine, Mount Sinai School of Medicine, New York, New York, USA.
Diabetes (Impact Factor: 8.1). 01/2012; 61(1):94-103. DOI: 10.2337/db11-0814
Source: PubMed

ABSTRACT

Growth hormone (GH) exerts diverse tissue-specific metabolic effects that are not revealed by global alteration of GH action. To study the direct metabolic effects of GH in the muscle, we specifically inactivated the growth hormone receptor (ghr) gene in postnatal mouse skeletal muscle using the Cre/loxP system (mGHRKO model). The metabolic state of the mGHRKO mice was characterized under lean and obese states. High-fat diet feeding in the mGHRKO mice was associated with reduced adiposity, improved insulin sensitivity, lower systemic inflammation, decreased muscle and hepatic triglyceride content, and greater energy expenditure compared with control mice. The obese mGHRKO mice also had an increased respiratory exchange ratio, suggesting increased carbohydrate utilization. GH-regulated suppressor of cytokine signaling-2 (socs2) expression was decreased in obese mGHRKO mice. Interestingly, muscles of both lean and obese mGHRKO mice demonstrated a higher interleukin-15 and lower myostatin expression relative to controls, indicating a possible mechanism whereby GHR signaling in muscle could affect liver and adipose tissue function. Thus, our study implicates skeletal muscle GHR signaling in mediating insulin resistance in obesity and, more importantly, reveals a novel role of muscle GHR signaling in facilitating cross-talk between muscle and other metabolic tissues.

Full-text

Available from: Gary J Schwartz
Targeted Loss of GHR Signaling in Mouse Skeletal
Muscle Protects Against High-Fat DietInduced
Metabolic Deterioration
Archana Vijayakumar,
1
YingJie Wu,
1
Hui Sun,
1
Xiaosong Li,
2
Zuha Jeddy,
1
Chengyu Liu,
3
Gary J. Schwartz,
2
Shoshana Yakar,
1
and Derek LeRoith
1
Growth hormone (GH) exerts diverse tissue-specic metabolic
effects that are not revealed by global alteration of GH action. To
study the direct metabolic effects of GH in the muscle, we
specically inactivated the growth hormone receptor (ghr)
gene in postnatal mouse skeletal muscle using the Cre/loxP
system (mGHRKO model). The metabolic state of the mGHRKO
mice was characterized under lean and obese states. High-fat
diet feeding in the mGHRKO mice was associated with reduced
adiposity, improved insulin sensitivity, lower systemic inam-
mation, decreased muscle and hepatic triglyceride content,
and greater energy expenditure compared with control mice. The
obese mGHRKO mice also had an increased respiratory exchange
ratio, suggesting increased carbohydrate utilization. GH-regulated
suppressor of cytokine signaling-2 (socs2) expression was de-
creased in obese mGHRKO mice. Interestingly, muscles of both lean
and obese mGHRKO mice demonstrated a higher interleukin-15
and lower myostatin expression relative to controls, indicating
a possible mechanism whereby GHR signaling in muscle could
affect liver and adipose tissue function. Thus, our study implicates
skeletal muscle GHR signaling in mediating insulin resistance in
obesity and, more importantly, reveals a novel role of muscle GHR
signaling in facilitating cross-talk between muscle and other met-
abolic tissues. Diabetes 61:94103, 2012
T
he metabolic actions of growth hormone (GH)
are largely attributed to the stimulation of lipol-
ysis, and the subsequent rise in free fatty acid
(FFA) ux from the adipose tissue resulting in
increased FFA uptake and use in the skeletal muscle
and liver (reviewed in 1,2). However, an inherent limitation
to studies using global alteration of GH action is the ac-
companying change in body com position as well as
changes in insulin-like growth factor (IGF)-1 and insulin
action, complicating our ability to dissect the tissue-specic
metabolic effects of GH.
An important aspect of the metabolic effects of GH is its
ability to antagonize insuli n action in the muscle, likely via
the direct stimulation of negative regulators of insulin
signaling such as suppressor of cytokine signaling (SOCS)
proteins and p85a regulatory subunit of phosphatidylino-
sitol 3-kinase (PI3K). SOCS proteins facilitate insulin re-
sistance by several mechanisms including mediation of
the proteasomal degradation of insulin receptor substrate
(IRS) proteins that are involved in insulin/IGF-1 signal
transduction (3). Excess GH levels have been associated
with increased skeletal muscle expression of p85a, which
can downregulate insulin signaling by preventing the ac-
tivation of the p110 catalytic subunit of PI3K (46). On the
other hand, GH-mediated increase in FFA ux into the
muscle and its su bsequent reesterication to trig lycerides
(TG) yield intermediates such as diacylglycerols and
ceramides that can attenuate insulin signaling by activa-
tion of protein kinase C (PKC) isoforms (7).
To address the muscle-specic effects of the growth hor-
mone receptor (GHR) on substrate metabolism and insulin
action, we generated the mGHRKO mouse model with in-
activation of ghr specically in postnatal skeletal muscle
using the Cre/loxP system. Our study implicates skeletal
muscle GHR signaling in inuencing substrate preference
and maintenance of whole-body energy homeostasis, par-
ticularly in the obese state.
RESEARCH DESIGN AND METHODS
The generation of the GHR
oxed
mice has been described elsewhere (8). The
GHR
oxed
mice were crossed with muscle creatine kinase (MCK-Cre) mice
[cat. no. 006475 B6.FVB (129S4)-Tg(Ckmm-Cre)5Khn/J; The Jackson Labora-
tory, Bar Harbor, ME] to derive the mGHRKO mice (9). The mice were housed
in the pathogen-free Association for the Assessment and Accreditation of
Laboratory Animal Care Internationalaccredited animal facilities of the
Mount Sinai School of Medicine and were kept on a 12-h light/dark cycle. All
experimental procedures were in accordance with the Institutional Animal
Care and Use Committee of the Mount Sinai School of Medicine. Male mice on
the C57BL/6 background were used for all experiments. The mice had ad
libitum access to water and either a standard laboratory diet (LabDiet,
Brentwood, MO) or a high-fat (60% fat) diet (HFD) (BioServ, New Brunswick,
NJ). In all experiments, the mGHRKO (GHR
ox/ox
; cre) mice were compared
with control mice (GHR
ox/ox
).
Semiquantitative PCR. Genomic DNA from various tissues of 7- to 8-wee k-
old mice was extracted using the conventional phenol-chloroform method.
DNA concentrations were determined using NanoDrop ND-1000 Spectropho-
tometer (Thermo Scientic, Wilmington, DE), and 500 ng DNA was subjected to
semiquantitative PCR using the primer set 59-CATTCTTTTCTGGGATGCTAT-39
and 59-CGGACATTGCATCTGTGATT-39, which detects the oxed, wild-type,
and null (recombinant) forms of the GHR.
In vivo stimulation. Mice were acutely stimulated with recombinant human
growth hormone (rhGH) (Genentech, San Francisco, CA), insulin (Humulin R;
Eli Lilly, Indianapolis, IN), IGF-1 (Genetech, San Francisco, CA), or 13 PBS,
anesthetized with 2.5% Avertin (2,2,2-Tribromoethanol dissolved in tert-amyl
alcohol; Sigma). The tissues were extracted, snap-frozen in liquid nitrogen,
and subjected to Western blot analysis.
From the
1
Division of Endocrinology, Diabetes, and Bone Diseases, the De-
partment of Medicine, Mount Sinai School of Medicine, New York, New
York; the
2
Departments of Medicine and Neuroscience, Albert Einstein Col-
lege of Medicine , Bronx, New York; and the
3
Transgenic Core Fac ili ty,
National Heart, Lung, and Blood Institute, National Institutes of Health,
Bethesda, Maryland.
Corresponding authors: Derek LeRoith, derek.leroith@mssm.edu, and Shoshana
Yakar, sy1007@nyu.edu.
Received 14 June 2011 and accepted 18 October 2011.
DOI: 10.2337/db11-0814
This article contains Supplementary Data online at http://diabetes
.diabetesjour nals.org /lookup/suppl /doi:10.23 37/db11-08 14/-/DC1.
A.V. and Y.W. contributed equally to this study.
Y.W., H.S., and S.Y. are currently afliated with the David B. Kriser Dental
Center, Department of Basic Science and Craniofacial Biology, New York
University College of Dentistry, New York, New York.
Ó 2012 by the American Diabetes Association. Readers may use this article as
long as the work is properly cited, the use is educational and not for prot,
and the work is not altered. See http://creativecommons.org/licenses/by
-nc-nd/3.0/ for details.
94 DIABETES, VOL. 61, JANUARY 2012 diabetes.diabetesjournals.org
ORIGINAL ARTICLE
Page 1
Growth parameters and circulating factors. Body composition analysis
was performed using the EchoMRI 3-in-1 NMR system (Echo Medical Systems,
Houston, TX). Body length was measured as nose-to-anus length at the time of
sacrice. Blood glucose level was measured from the tail using an automated
glucometer (Elite; Bayer, Mishawaka, IN). Serum or plasma collected from the
tail vein or retro-orbital sinus was used for measurement of circulating insulin
(Linco, St. Charles, MO), IGF-1 (Alpco, Salem, NH), GH (Millipore, Bellirica, MA),
FFA (Roche Applied Science, India napolis , IN), TG (Pointe Scientic, Canton,
MI), interleukin (IL)-6, and adiponectin (R&D Systems, Minneapolis, MN) per the
manufacturers instructions.
Food intake, glucose and insulin tolerance tests, and hyperinsulinemic-
euglycemic clamp. Food intake was measured weekly as previously published
(10). Glucose and insulin tolerance tests were performed in unrestrained mice as
previously described (10). Hyperinsulinemic-euglycemic clamps were performed
in unrestrained mice fed HFD for 1420 weeks as previously described (11).
Histology and immunoourescence. Liver and epidydimal fat pad were xed
in 10% PBS-buffered formalin; parafn sections were processed and subjected
to either hematoxylin-eosin (H-E) staining or immunoorescence as previously
described (12). For the immunoourescence, F4/80 primary antibody (Abcam,
Cambridge, MA) with Alexa Fluor goat anti-rat secondary antibody (Molecular
Probes, Eugene, OR) was used. Pictures were obtained using the Olympus
AX70 camera (Olympus, Center Valley, PA) at 103 objective magnication.
ImageJ software (NIH, Bethesda, MD) was used to quantify the cross-sectional
area of at least 150250 adipocytes and fraction of the F4/80
+
area.
Western blot. Protein extraction from the liver, quadriceps, and epidydimal fat
pad and Western blot analyses were performed as previously described (10).
The primary antibodies used were phosphorylated signal transducer and acti-
vator of transcription (Stat)5
Tyr694
, phosphorylated IGF-1 receptor b
Tyr1135/1136
/
insulin receptor b
Tyr1150/1151
, phosphorylated Akt
Ser473
, Akt, phosphorylated
p42/44 MA P ki nase
Thr202/Tyr204
(phosphorylated extracellular signalrelated
kinase [Erk]), and p42/44 MAP kinase (Erk), phosphorylated AMP-activated
protein kinase (AMPK)a
Thr172
, and AMPKa from Cell Signaling Technology
(Danvers, MA); STAT5, IRb, IGF-IRb, CD36, p85a,andb-tubulin from Santa
Cruz Biotechnology (Santa Cruz, CA); and lipoprotein lipase (LPL) and SOCS-3
from Abca m (Cambridge, MA). Anti-rabbi t secondary antibody from Li-cor
Biosciences (Lincoln , NE) was us ed. The blots w ere quantied using the
Odyssey Infrar ed Imagin g System Appl ication softwa re (version 3.0; Li-cor
Biosciences).
Gene expression analyses. Total RNA from quadriceps was extracted using
TRIzol reagent and reverse transcribed according to the manufa cturers
instructions (Invitrogen, Carlsbad, CA). Real time-PCR was performed using
the QuantiTect SYBR green PCR kit (QIAGEN, Valencia, CA) in ABI PRISM
7900HT sequence detection systems (Applied Biosy stems, Foster City, CA).
The primers are listed in Supplementary Table 1; for each gene, a single
sample was assaye d three times and gene e xpression was normalized to
gapdh.
Tissue TG and indirect calorimetry. Total TG were extracted from the liver
and gastrocnemius using the chloroform-methanol method (13). TG content
was quantied using TG reagent (Pointe Scientic, Canton, MI). Metabolic cage
studies were performed in unrestrained mice fed an HFD for 1214 weeks as
previously described (11).
Statistical analysis. All results are expressed as means 6 SEM. One-way (for
comparison of two groups) or two-way ANOVA (for comparison of three or
more groups) with Holm Sidak post hoc test was performed using SigmaStat
for Windows (version 3.5; Systat Software, Inc., Chicago, IL).
RESULTS
mGHRKO mice harbor skeletal musclespecic ghr
gene inactivation. Skeletal musclespecic ghr gene
recombination in the mGHRKO mice was validated at the
level of genomic DNA and mRNA expression (Fig. 1A and
B). Acute rhGH (125 mg/kg i.p. for 15 min) stimulation
elicited robu st STAT5 phosphorylation in the muscle of
control mice but not mGHRKO mice, while the livers of
both control and mGHRKO mice displayed a similar re-
sponse (Fig. 1C). Interestingly, los s of ghr in postnatal
skeletal muscle did not affect muscle igf-1 expression in
the mGHRKO mice, and mice did not respond differently
to an ac ute IGF-1 stimula tion (1 mg/kg i.p. for 5 min)
(Fig. 1B and Supplementary Fig. 1E). Moreover, circu-
lating IGF-1 and GH levels and body length were similar
between control and mGHRKO mice at 16 weeks of age
(Table 1).
mGHRKO mice are protected against HFD-induced
insulin resistance. The lean mGHRKO mice displayed
reduced body weight in adulthood, starting at 7 weeks of
age (Supplementary Fig. 1A). HFD feeding starting at 57
weeks of age increased body weights of both the con trol
and mGHRKO mice relative to the lean mice; however, the
obese mGHRKO mice were signicantly lighter than the
obese control mice (Fig. 2A). Absolute lean mass was
signicantly decreased in both lean and obese mGHRKO
mice compared with the control mice (Fig. 2B). However,
while the lean mGHRKO mice had lower absolute fat mass
than the lean control mice (Supplementary Fig. 1B), rela-
tive body adiposity was signicantly reduced only in the
obese mGHRKO mice compared with the obese control
mice (Fig. 2C). The lower body weight and fat mass in the
obese mGHRKO mice could not be explained by lower
food intake, which was similar in both groups (Supple-
mentary Fig. 2A). Moreover, wet tissue weights of the
gonadal and subcutaneous fat pads were also signi-
cantly decreased in the lean and obese mGHRKO mice
compared with thei r respective controls (Table 1). The
obese mGHRKO mice demonstrated lower circulating
FIG. 1. mGHRKO mice have skeletal musclespecic ablation of the ghr
gene. A: Genomic DNA was isolated from the indicated tissues of 7- to
8-week-old control and mGHRKO mice, and recombination of ghr was
assessed by semiquantitative PCR. The recombinant null ghr is
expressed only in the various muscle groups and heart of the mGHRKO
mice. n =23/group. (Representative images are shown.) Quad, quad-
riceps; EDL, extensor digitorum longus; Sm. Intes, small intestine. B:
Real-time PCR analysis of ghr and igf-1 expression in quadriceps of
16-week-old control and mGHRKO mice. Gene expression was normal-
ized to gapdh (n =34/group). C: Acute GH stimulation (125 mg/kg
body wt i.p. for 15 min) of 7- to 8-week-old mice and probing for STAT5
phosphorylation in the liver and quadriceps by immunoblot analysis.
Phosphorylated levels of STAT5 (pSTAT5) were normalized to total
STAT5 levels (n =24/group). *P £ 0.05, one-way ANOVA.
A. VIJAYAKUMAR AND ASSOCIA TES
diabetes.diabetesjournals.org DIABETES, VOL. 61, JANUARY 2012 95
Page 2
TG levels compared with the obese controls, and while the
lean mGHRKO mice had reduced circulating FFA levels
compared with controls, no difference was observed in
the obese mice (Table 1). Like the lean mice, the obese
control, but not obese mGHRKO, mice displayed robust
muscle STAT5 phosphorylation in response to acute rhGH
stimulation (125 mg/kg i.v. for 10 min), while the hepatic re-
sponse was similar between the two groups (Supplementary
Fig. 2B).
We next sought to determine whether the reduced body
weight and fat mass in the lean and obese mG HRKO mice
translated into better metabolic outcomes. Despite signif-
icantly lower fed-state blood glucose levels (Table 1), the
lean mGHRKO mice did differ from the lean controls in
their serum insulin levels or response to a gluco se toler-
ance test, an insulin tolerance test, or acu te insulin stim-
ulation (Fig. 2D and Supplementary Fig. 1CE). However,
the obese mGHRKO mice had signicantly lower insulin
levels than did the obese control mice (Fig. 2D)anddem-
onstrated improved insulin sensitivity during glucose toler-
ance and insulin tolerance tests (Fig. 2E and F).
Consequently, the obese mG HRKO and control mice
were subjected to hyperinsulinemic-euglycemic clamps to
determine which tissue(s) contributed to the improved in-
sulin sensitivity. The obese mGHRKO mice demonstrated
improved hepatic insulin sensitivity, as evidenced by higher
glucose infusion rate (GIR) and greater extent of insulin-
mediated suppression of hepatic glucose production (HGP)
(Fig. 3A and B). The obese mGHRKO mice also had an in-
creased rate of whole-body glucose disposal (R
d
), with
a twofold increase in insulin-stimulated muscle and white
adipose tissue glucose uptake compared with the control
mice during the clamp period (Fig. 3CE). Furthermore,
Akt phosphorylation in response to acute insulin stimu-
lation (1 unit/kg i.p. for 5 min) was signicantly greater or
tended to be higher in the adipose tissue and skeletal
muscle, respectively, of the obese mGHRKO mice relative
to the obese cont rols (Fig. 3F and G). Nonetheless, p85a
protein content, which inversely correlates with insulin
sensitivity, did not differ between the muscles of the obese
control and mGHRKO mice (Supplementary Fig. 2C).
The reduced fat mass in the obese mGHRKO mice was
associated with smaller a dipocytes compared with the
obese controls (Fig. 4A). Furthermore, the obese mGHRKO
mice displayed signicantly lower circulating IL-6 levels as
well as adipose tissue macrophage inltration, as assessed
by F/480 staining, suggesting reduced obesity-associ ated
inammation (Table 1 and Fig. 4B). Interestingly, the lean
mGHRKO mice displayed signicantly higher serum adi-
ponectin levels compared with the lean control mice;
however, there was no difference in adiponectin levels in
the obese mice (Table 1). Lean mGHRKO mice had similar
liver and muscle TG content compared to lean controls.
However, while HFD feeding increased tissue TG content
in both groups, the obese mGHRKO mice had signicantly
less liver and muscle TG content than did the obese con-
trols (Fig. 4C and E). In concordance with this, the obese
control but not obese mGHRKO mice developed marked
hepatic steatosis (Fig. 4D).
Obese mGHRKO mice demonstrate improved meta-
bolic efciency. The reduced body adiposity in the obese
mGHRKO mice was associated with improved insulin
sensitivity, which may result from increased metabolic
efciency (analyzed by indirect calorimetry). As hypothe-
sized, the obese mGHRKO mice showed increased light-
and dark-phase oxygen consumption (VO
2
)(Fig.5A)and
CO
2
production (VCO
2
) (Fig. 5B), resulting in a higher re-
spiratory exchange ratio (Fig. 5C) suggestive of greater
carbohydrate utilization. The obese mGHRKO mice also
demonstrated greater energy expenditure in both the light
and the dark phases (Fig. 5D). The mGHRKO mice had
increased locomot or activity, which may have further
contributed to their increased energy expenditure (data not
shown).
Skeletal muscle lipid metabolism is improved in obese
mGHRKO mice relative to obese controls. GH has
known roles in lipid uptake and metabolism, and the obese
mGHRKO mice also displayed lower muscle TG content.
We subsequently found a reduction in the expression of
genes involved in de novo lipogenesis and TG reester-
ication such as fatty acid synthase (fasn), sterol regulatory
binding protein-1 (srebp-1), and diglyceride acyltransferase-1
(dgat1) in the obese mGHRKO muscles relative to controls
(Fig. 6A). There was no difference in the expression of
b-oxidation markers such as carnitine palmitoyltransfer-
ase 1-a (cpt1a) and peroxiso me proliferatoractivated
receptor g coactivator 1-a (pgc1a)(Fig.6A). Addition-
ally, protein content of LPL and the fatty acid transporter
CD36, which are involved in fatty acid uptake, did not
differ between the obese control and mGHRKO mice
(Supplementary Fig. 2 C).
Altered expression of muscle markers in mGHRKO
mice that could contribute to their insulin sensitivity.
SOCS proteins are negative regulators of insulin signaling,
and we found a red uction in the mRNA expression of socs2
but not socs1 in the obese mGHRKO mice (Fig. 6B). There
TABLE 1
Characterization of lean and obese control and mGHRKO mice
Lean Obese
Control mGHRKO Control mGHRKO
Body length, n =410 (cm) 9.91 6 0.04 10.08 6 0.08 10.25 6 0.16 10.02 6 0.16
GH, n =67 (ng/mL) 11.84 6 8.38 9.24 6 5.11 n.d. n.d.
IGF-1, n =710 (ng/mL) 316.29 6 6.27 309.50 6 8.90 n.d. n.d.
Fed state blood glucose, n =1833 (mg/dL) 150.04 6 4.50 129.73 6 3.36* 163.05 6 6.22 158.78 6 5.76
Fed state FFA, n =915 (nmol/mL) 0.40 6 0.03 0.30 6 0.08* 0.32 6 0.02 0.39 6 0.04
Gonadal fat pad weight, n =1427 (g) 0.596 6 0.04 0.446 6 0.03* 2.93 6 0.14 2.34 6 0.08*
Subcutaneous fat pad weight, n =727 (g) 0.319 6 0.02 0.229 6 0.02* 1.95 6 0.22 1.21 6 0.07*
IL-6, n =429 (pg/mL) 6.25 6 2.33 13.57 6 4.07 19.80 6 4.13 9.91 6
1.56*
Adiponectin, n =441 (ng/mL) 2.87 6 0.32 3.83 6 0.08* 4.59 6 0.21 4.73 6 0.17
Data are means 6 SEM. n.d., not determined. *P # 0.05 control vs. mGHRKO, one-way ANOVA.
MUSCLE GHR SIGNALING AND ENERGY HOMEOSTASIS
96 DIABETES, VOL. 61, JANUARY 2012 diabetes.diabetesjournals.org
Page 3
FIG. 2. Obese mGHRKO mice have reduced adiposity and improved insulin sensitivity. Control and mGHRKO mice were placed on an HFD (60%
of calories from fat) starting at 57 weeks of age and were compared with littermat es fed a standard laboratory diet. A: Body weight. B: Lean
body mass. C: Relative body adiposity. D: Circulating insulin levels of lean and obese control and mGHRKO mice were measured at indicated times
(n =918/group). Glucose tolerance (1 g/kg) (E) and insulin tolerance (0.5 units/kg) (F) tests in mice fed an HFD for 22 weeks (n = 10/group). All
values are represented as means 6 SEM.
a
P £ 0.05 for obese control vs. obese mGHRKO,
b
P £ 0.05 for lean control vs. lean mGHRKO,
c
P £ 0.05 for
lean control vs. obese control,
d
P £ 0.05 for lean mGHRKO vs. obese mGHRKO; one- and two-way ANOVA.
A. VIJAYAKUMAR AND ASSOCIA TES
diabetes.diabetesjournals.org DIABETES, VOL. 61, JANUARY 2012 97
Page 4
FIG. 3. Improved insulin sensitivity in obese mGHRKO mice as assessed by hyperinsulinemic-euglycemic clamps. Mice were placed on an HFD (60% of
calories from fat) starting at 57 weeks of age and were subjected to hyperinsulinemic-euglycemic clamps after 1214 weeks of HFD feeding. A: GIR.
B: HGP at basal and insulin-stimulated states (left panel) and extent of insulin-mediated suppression of HGP (right panel). C:Whole-bodyR
d
. D:
Skeletal muscle R
d
. E: Adipose tissue R
d
(fat R
d
) at the end of the clamp period was measured (n = 5/group for all clamp studies). F and G:Im-
munoblot analysis of white adipose tissue (F) and skeletal muscle (G) response to an acute insulin (1 unit/kg i.p. for 5 min) stimulation of mice fed an
HFD as evaluated by phosphorylation of insulin receptor (pIR), Akt (pAkt), and Erk (pErk), which are normalized to total protein levels (n =34
/group). The blots were quantied and represented as the extent of stimulation with respect to PBS-treated levels for each group. All values are
represented as means 6 SEM. *P £ 0.05, one-way ANOVA.
MUSCLE GHR SIGNALING AND ENERGY HOMEOSTASIS
98 DIABETES, VOL. 61, JANUARY 2012 diabetes.diabetesjournals.org
Page 5
FIG. 4. mGHRKO mice are protected from complications of HFD-induced insulin resistance and obesity. Mice were placed on an HFD (60% of
calories from fat) starting at 57 weeks of age for a period of 14 weeks and, where indicated, were compared with lean mice. A: Adipocyte area
as evaluated by H-E staining (right panel) and the area of 150200 adipocytes were quantied using the NIH ImageJ software (left panel)(n =
7/group). (Representative images are shown.) B: Immunoourescent staining for F4/80
+
adipocytes in epidydimal adipose tissue; the fraction of
F4/80
+
area was quantied using the NIH ImageJ software (n = 7/group). (Representative images are shown.) C: Liver TG content (n =611/
group). D: Hepatic steatosis as evaluated by H-E staining (n =67/group). (Representative images are shown.) E: Muscle TG content determined in
the gastrocnemius (n =611/group). In all of the images, the scale bar represents 200 mm. All values are represented as means 6 SEM.
a
P £ 0.05 for
obese control vs. obese mGHRKO,
b
P £ 0.05 for lean control vs. lean mGHRKO,
c
P £ 0.05 for lean control vs. obese control,
d
P £ 0.05 for lean
mGHRKO vs. obese mGHRKO; one- and two-way ANOVA. (A high-quality color representation of this gure is available in the online issue.)
A. VIJAYAKUMAR AND ASSOCIA TES
diabetes.diabetesjournals.org DIABETES, VOL. 61, JANUARY 2012 99
Page 6
was also no difference in protein content of SOCS3 in the
obese control and mGHRKO mice (Supplementary Fig. 2C).
Additionally, the expression of myostatin (mstn) and
il-15 was reduced and increased, respectively, in the skel-
etal muscles of the mGHRKO mice (Fig. 6B). There was no
difference in AMPK phosphorylation between the lean and
obese mice of both groups (data not shown).
DISCUSSION
Herein, we report that loss of skeletal muscle GHR signaling
improves insulin sensitivity in the context of diet-induced
obesity. While HFD f eeding worsened the metabolic state
of both control and mGHRKO mice compared with their
lean counterparts, the obese mGHRKO mice demonstrated
signicantly lower body weight, body adiposity, and insulin
resistance relative to the obese controls. Hyperinsulinemic-
euglycemic clamp studies indicated increased GIR and
muscle and adipose tissue R
d
in obese mGHRKO mice
compared with obese controls. These data were substanti-
ated with greater insulin-stimulated Akt phosphorylation in
the white adipose tissue and muscle of the obese mGHRKO
mice. Further, indirect calorimetry studies revealed greater
energy expenditure, which was associated with greater
carbohydrate utilization in the obese mGHRKO mice.
The improved insulin sensitivity of the obese mGHRKO
mice could have arisen as a consequence of direct GH
FIG. 5. Obese mGHRKO mice are more metabolically efcient than
obese controls. Mice were placed on an HFD (60% of calories from
fat) starting at 57 weeks of age for a period of 14 weeks, and their
thermodynamic properties were studied in m etabolic cages. A:Oxy-
gen consumption (VO
2
). B:CO
2
production (VCO
2
). C: R espiratory
exchange rat io (RER). D: Energy expenditure (EE) (n =8/group).
All values are represented as means 6 SEM. *P £ 0.05, one-way
ANOVA.
FIG. 6. Altered gene expression in the obese mGHRKO muscles. Mice
were placed on an HFD (60% of calories from fat) starting at 57 weeks
of age for a period of 14 weeks, and the quadriceps muscles were ana-
lyzed. A: Real-time PCR analysis of indicated genes is represented as
fold change compared with expression of obese control mice. Gene
expression was normalized to gapdh (n =45/group). B: Expression of
socs2, socs1, mstn,andil-15 in muscles of lean and obese control and
mGHRKO mice was quantied by real-time PCR, normalized to gapdh,
and represented as fold change compared with expression in lean
control mice (n =35/group). The double line on the y-axis denotes
where the scale was broken. All values are represented as means 6
SEM.
a
P £ 0.05 for obese control vs. obese mGHRKO,
b
P £ 0.05 for lean
control vs. lean mGHRKO; one- and two-way ANOVA.
MUSCLE GHR SIGNALING AND ENERGY HOMEOSTASIS
100 DIABETES, VOL. 61, JANUARY 2012 diabetes.diabetesjournals.org
Page 7
action in the skeletal muscle or indirectly as a result of
improvements in other tissues such as the liver and adipose
tissue. As we discuss below, our data with the mGHRKO
mice provide a rationale for both possibilities.
The obese mGHRKO mice demonstrate increased insulin-
stimulated skeletal muscle glucose uptake and a shift in
substrate utilization toward increased carbohydrate me-
tabolism. Moreover, the obese mGHRKO mice had lower
muscle TG content, which could reect either reduced
lipid uptake into the skelet al muscle or increased lipid
oxidation. GH has been shown to reduce LPL activity in
the adipose tissue and, thus, suppress adipose tissue lipid
uptake, but its effect on muscle LPL activity is not well
characterized (1417). Accordingly, we did not nd a dif-
ference in LPL protein content in the obese mGHRKO mice
compared with the obese controls. The protein content
of the fatty acid transporter CD36, which is also regulated
by subcellular localization (18,19), was also not different
between the obese control and mGHRKO mice. Fu r t h e r -
more, we found reduced expression of genes involved
in de novo lipogenesis and TG reestericationinthe
obese mGHRKO muscles, while expression of genes in-
volved in b-oxidation was not different. Nevertheless,
protein content or mRNA expression does not reect
substrate ux, which could be different between the
obese mGHRKO and co ntrol muscles. Muscle TG content
i s inversely corr elated with insulin sensitivity because
of accumulation of intermediates such as diacylglycerols
and ceramides that can inhibit insulin signaling by activa-
tion of PKC isoforms (7). Thus, lower muscle TG content in
the obese mGHRKO mice could potentially account for
enhanced muscle insulin sensitivity in the face of HFD
feeding.
GH can interfere with insulin signaling by inducing the
expression of SOCS proteins, especially SOCS2 and SOCS3
(3,20,21). Accordingly, muscles of obese mGHRKO mice
had decreased socs2 but not socs1 mRNA expression.
Thus, lower socs2 expression could, to some degree, ex-
plain the increased insulin action in the mGHRKO muscles.
The p85a regulatory subunit of PI3K is regulated by GH
and is overexpressed in the insulin-resistant muscle, where
it is hypothesized to bind to and sequester IRS-1, thereby
preventing the activation of the p110 catalytic subunit of
PI3K. While total protein content of p85a did not differ
between the obese control and mGHRKO mice, we could
not determine whether the p85a levels that we detected
represent its monomeric or homo/hetero-dimeric forms in
the obese control versus mGHRKO muscles.
A recent study by Mavalli et al. (22) reported that
knockout of the GHR in skeletal muscle using the myocyte-
specic enhancer factor 2C promoter resulted in enhanced
body weight, body adiposity, and circulating TG levels and
worsened insulin sensitivity. While the knockout mice
demonstrated decreased dark-phase locomotor activity,
they tended to have increased energy expenditure. More-
over, primary myotubes isolated from the knockout mice
demonstrated decreased insulin-stimulated glucose uptake,
insulin receptor protein content, and basal Akt ( Thr 308
but not Ser 473) and Erk phosphorylation. Interestingly,
primary myoblast cultures isolated from t he knockout
mice displayed reduced inhibitory Ser phosphorylation
of IRS-1 at residues 612 and 636/639, while Ser phosphor-
ylation of residue 1101, which is regulated by the diac-
ylglycerol-responsive PKCu, was higher (23). These data are
contrary to our observations in the mGHRKO mice, which
had reduced fat mass and slightly improved metabolism
under lean conditions and were protected from HFD-induced
insulin resistance. Moreover, we did not observe any dif-
ference in insulin receptor content or basal Akt or Erk
phosphorylation in who le-muscle tissue lysates of the lean
mGHRKO mice.
The discrepancies between the two studies may arise
from the different genetic background of the mice used;
the strain-specic differences in metabolic response are
well documented (24). Additionally, the MCK promot-
er used to drive the Cre recombinase expression in the
mGHRKO mice is expressed prim arily in postnatal skeletal
and cardiac muscle (25). The myocyte-specic enhancer
factor 2C promoter, while expressed only in the skeletal
muscle postnatally (26), is also expressed prenatally at 7.5
days postconception in the heart and regions that develop
into the brain (27). Thus, it is conceivable that changes
arising from early inactivation of the GHR in the de-
veloping somite may inuence the phenotype of the mice
in adulthood. Further, the molecular data presente d by
Mavalli et al. were generated in an in vitro system using
primary myoblasts isolated at 4 weeks of age, when the
knockout mice do not demonstrate an insulin-resistant phe-
notype in vivo. On the other hand, our experiments were
performed in whole tissue lysates isolated at either 16 weeks
of age or 14 weeks of HFD feeding, when the mGHRKO
mice showed a clear phenotype.
Inactivation of stat5a/b in the skeletal muscle using the
Myf5 promoter to drive Cre expression resulted in reduced
body length and body weight (primarily due to a reduction
in lean mass) and worsened glucose tolerance (28). How-
ever, similar to the above-mentioned study, these mice
were on a mixed genetic background and the Myf5 promoter
was also expressed prenatally at 8 days postconception
(29). Moreover, while GH is one of the major activators of
STAT5, other factors, including insulin, can also induce
this transcription factor (30,31); furthermore, GH does not
signal exclusively through STAT5 (32).
An interesting point of comparison between our obser-
vations and the two aforementioned studies is muscle igf-1
expression in the knockout mice. While we did not ob-
serve a difference in igf-1 expression in adult mGHRKO
mice, both of the other studies reported a signicant re-
duction in muscle igf-1 expression. Moreover, the muscle-
stat5 a/b knockout mice also demonstrated a 15% reduction
in circulating IGF-1 levels. This suggests that muscle igf-1
expression is regulated by GH prenatally but not post-
natally. Indeed, studies performed in both in vitro and in
vivo settings have reported GH-independent changes in
expression of the different IGF-1 isoforms under various
experimental conditions (3336).
Another signicant difference between the ndings of
the study by Mavalli et al. and those in the mGHRKO mice
is that while the former reporte d increased body adiposity,
we observed the opposite in the mGHRKO mice. Thus, it is
very possible that the difference in adiposity may be respon-
sible for the difference in insulin sensitivity between the two
models. Moreover, the obese mGHRKO mice also demon-
strated increased adipose tissue and hepatic insulin sensitiv-
ity in the hyperinsulinemic-euglycemi c clamp studies, as well
as reduced adipose tissue macrophage inltration, liver TG
content, and circulating TG levels. The obese mGHRKO
mice had signicant reductions in circulating IL- 6 l ev els,
an adipocytokine associated with obesity-induced systemic
inammation insulin resistance (37). Circulating adiponectin
levels, which positively correlate with insulin sensitivity (37)
and may inuence muscle metabolism by promoting the
A. VIJAYAKUMAR AND ASSOCIA TES
diabetes.diabetesjournals.org DIABETES, VOL. 61, JANUARY 2012 101
Page 8
deacetylation of PGC-1a (38), were increased in the lean
but not obese mGHRKO mice. All these factors could have
a major impact on the insulin sensitivity of the obese
mGHRKO mice.
An interesting implication of our study is that GH fa-
cilitates tissue cross-talk, possibly via the regulation of
secreted myokines that affect adipose tissue and liver
metabolism. Indeed, we found a reduction in the exp res-
sion of mstn in the mGHRKO mice. Whole-body and
skeletal musclespecic myostatin knockout mice dem-
onstrated improved insulin sensitivity and protection from
HFD-induced obesity and insulin resistance (3941). The
improved insulin sensitivity in the myostatin knockout
mice was attributed to increased AMPK phosphorylation
and energy expenditure (40,42). However, while the obese
mGHRKO mice displayed increased energy expenditure
we could not detect an increase in AMPK phosphorylation
in the lean or obese mGHRKO mice. Further, Oldham et al.
(43) reported that GH may regulate myostatin mRNA and
protein expression via STAT5-dependent and -independent
mechanisms. We also observed an increase in il-15 ex-
pression in the lean and obese mGHRKO mice. Unlike
myostatin, IL-15 positively affects metabolism by mod-
ulating substrate metabolism in the liver, white adipose
tissue, and brown adipose tissue (44). While we had
technical difculties in validating our mRNA expression
levels at the protein level, these preliminary ndings nev-
ertheless provide exciting new areas for future research.
Obesity is often described as a GH-suppressed state.
Given this, it is paradoxical that we observed strong
metabolic improvements in the obese mGHRKO mice.
Mean peak GH levels, GH pulse amplitude, and the rate of
GH secretion (but not the frequency of GH pulses) have
been shown to be reduced in obese individuals (45,46).
Further, obese, insulin-resistant mice are responsive to an
acute GH stimulation. Likewise, obese women treated with
GH respond in a similar manner to nonobese women in
terms of fat loss and the suppression of adipose tissue LPL
activity (18). Thus, obesity is associat ed with reduced but
not absent GH secretion, and the metabolic action of GH
may still persist in this state.
In conclusion, through use of mGHRKO mice we de-
scribe roles for muscle GHR signaling in mediating whole-
body insulin resistance in obesity. This may occur via
alterations in substrate utilization and energy expendi-
ture. An interesting implication of our study is the effect
that loss of GHR signaling in the muscle has on other
metabolic tissues, such as the adipose tissue and liver,
which bears further investigation.
ACKNOWLEDGMENTS
X.L. and G.J.S. are supported by the NIH DK-20541 grant
from the National Institutes of Health.
No potential conicts of interest relevant to this article
were reported.
A.V. designed and performed the experiments and wrote
the manuscript. Y.W. designed and performed the ex-
periments and reviewed and edited the manuscript. H.S.,
X.L., Z.J., and C .L. performed the experiments . G.J.S. and
S.Y. designed the experiments, reviewed and edited the
manuscript, and contributed to discussion. D.L. designed
the experiments, reviewed and edited the man uscript,
contributed to discussion, and is the guarantor.
The authors thank Caroline Kornhauser, Yosef
Chodakiewitz, Darren Gorman, Joshua Vazhapilly, Dr. Emily
Gallagher, and Dr. Rosalyn Ferguson from the Mount Sinai
School of Medicine for their assistance with the experi-
ments and review of the manuscript.
Parts of this study were presented in abstract form at the
93rd Annual Meeting of The Endocrine Society, Boston, MA,
47 June 2011, and the 5th International Congress of the
GRS and IGF Society, New York, NY, 37 October 2010.
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  • Source
    • "Standard rat fodder [license No. SCXK (Guangdong Province, China) 2003-0002; Guangdong supervison No. 2008D002] was provided by the Medical Laboratory Animal Center of Guangdong Province in China. Pregnant rats subjected to dietary restriction were utilized to establish the SGA rat model [17]. Rats were randomly housed in standard rat cages at a 2∶1 female-to-male ratio. "
    [Show abstract] [Hide abstract] ABSTRACT: Objective Insulin resistance has been observed in individuals born small for gestational age (SGA) with catch-up growth (CUG), yet the mechanisms involved remain unclear. This study examined the role of GH and insulin signaling crosstalk in insulin resistance of SGA rats with CUG. Design and Methods SGA rats were developed by dietary restriction in pregnant rats. GH receptor inhibition was performed on four-week old CUG-SGA and AGA rats. Phosphorylation of IRS-1, AKT, and ERK, and expression of SOCS3 in the skeletal muscle were determined via immunoblot analysis at baseline and after insulin stimulation in CUG-SGA, NCUG-SGA and AGA groups. Results Compared to AGA controls, phosphorylation of IRS-1 and AKT in response to insulin stimulation in CUG-SGA rats was significantly blunted (P<0.05), and phosphorylation of ERK at baseline was dramatically activated (P<0.05). SOCS3 expression was significantly increased in CUG-SGA compared to AGA (P = 0.001) and NCUG-SGA (P = 0.006) rats, and was significantly suppressed following GHR inhibition (P<0.05). Furthermore, phosphorylation of IRS-1 and AKT in response to insulin stimulation increased after GHR inhibition (P<0.05). Conclusions Insulin resistance in CUG-SGA rats is associated with impairment of IRS-1-PI3K-AKT signaling, which may result from GH signaling-induced up-regulation of SOCS3.
    Full-text · Article · Jun 2014 · PLoS ONE
  • Source
    • "Insulin resistance in skeletal muscle has long been recognized as a characteristic feature of type 2 diabetes and plays a major role in the pathogenesis of the disease [1]. Although several epidemiological data have shown that the consumption of added sugars as ingredients in processed or prepared foods and caloric beverages has dramatically increased over the last decades, most of the experimental studies investigating the development of insulin resistance in the skeletal muscle have been based on genetic manipulation or use of high fat diets234. In contrast, the molecular mechanisms underlying the detrimental effects of sugar, mainly those on skeletal muscle, are not completely understood. "
    [Show abstract] [Hide abstract] ABSTRACT: Peroxisome Proliferator Activated Receptor (PPAR)- δ agonists may serve for treating metabolic diseases. However, the effects of PPAR- δ agonism within the skeletal muscle, which plays a key role in whole-body glucose metabolism, remain unclear. This study aimed to investigate the signaling pathways activated in the gastrocnemius muscle by chronic administration of the selective PPAR- δ agonist, GW0742 (1 mg/kg/day for 16 weeks), in male C57Bl6/J mice treated for 30 weeks with high-fructose corn syrup (HFCS), the major sweetener in foods and soft-drinks (15% wt/vol in drinking water). Mice fed with the HFCS diet exhibited hyperlipidemia, hyperinsulinemia, hyperleptinemia, and hypoadiponectinemia. In the gastrocnemius muscle, HFCS impaired insulin and AMP-activated protein kinase signaling pathways and reduced GLUT-4 and GLUT-5 expression and membrane translocation. GW0742 administration induced PPAR- δ upregulation and improvement in glucose and lipid metabolism. Diet-induced activation of nuclear factor-κB and expression of inducible-nitric-oxide-synthase and intercellular-adhesion-molecule-1 were attenuated by drug treatment. These effects were accompanied by reduction in the serum concentration of interleukin-6 and increase in muscular expression of fibroblast growth factor-21. Overall, here we show that PPAR- δ activation protects the skeletal muscle against the metabolic abnormalities caused by chronic HFCS exposure by affecting multiple levels of the insulin and inflammatory cascades.
    Full-text · Article · Jun 2013 · Mediators of Inflammation
  • Source
    • "We have previously shown that loss of GHR signaling in the skeletal muscle in the mGHRKO mice is associated with a marked protection from the development of HFD-induced insulin resistance, as assessed by several parameters. In addition, the HFD-fed mGHRKO mice had a higher RER in the light phase under conditions of ad libitum feeding, indicative of greater whole-body carbohydrate utilization [8]. This indicated that lipid oxidation is suppressed in the mGHRKO mice, and we explored this hypothesis in this study. "
    [Show abstract] [Hide abstract] ABSTRACT: Growth hormone (GH) stimulates whole-body lipid oxidation, but its regulation of muscle lipid oxidation is not clearly defined. Mice with a skeletal muscle-specific knockout of the GH receptor (mGHRKO model) are protected from high fat diet (HFD)-induced insulin resistance and display increased whole-body carbohydrate utilization. In this study we used the mGRHKO mice to investigate the role of muscle GHR signaling on lipid oxidation under regular chow (RC)- and HFD- fed conditions, and in response to fasting. Expression of lipid oxidation genes was analyzed by real-time PCR in the muscles of RC- and HFD- fed mice, and after 24 h fasting in the HFD-fed mice. Expression of lipid oxidation genes was lower in the muscles of the mGHRKO mice relative to the controls, irrespective of diet. However, in response to 24 h fasting, the HFD-fed mGHRKO mice displayed up-regulation of lipid oxidation genes similar to the fasted controls. When subjected to treadmill running challenge, the HFD-fed mGHRKO mice demonstrated increased whole-body lipid utilization. Additionally, under fasted conditions, the adipose tissue of the mGHRKO mice displayed increased lipolysis as compared to both the fed mGHRKO as well as the fasted control mice. Our data show that muscle GHR signaling regulates basal lipid oxidation, but not the induction of lipid oxidation in response to fasting. We further demonstrate that muscle GHR signaling is involved in muscle-adipose tissue cross-talk; however the mechanisms mediating this remain to be elucidated.
    Full-text · Article · Sep 2012 · PLoS ONE
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