Diet-induced muscle insulin resistance is associated with extracellular matrix remodeling and interaction with integrin alpha2beta1 in mice.

Abstract

The hypothesis that high-fat (HF) feeding causes skeletal muscle extracellular matrix (ECM) remodeling in C57BL/6J mice and that this remodeling contributes to diet-induced muscle insulin resistance (IR) through the collagen receptor integrin α(2)β(1) was tested.
The association between IR and ECM remodeling was studied in mice fed chow or HF diet. Specific genetic and pharmacological murine models were used to study effects of HF feeding on ECM in the absence of IR. The role of ECM-integrin interaction in IR was studied using hyperinsulinemic-euglycemic clamps on integrin α(2)β(1)-null (itga2(-/-)), integrin α(1)β(1)-null (itga1(-/-)), and wild-type littermate mice fed chow or HF. Integrin α(2)β(1) and integrin α(1)β(1) signaling pathways have opposing actions.
HF-fed mice had IR and increased muscle collagen (Col) III and ColIV protein; the former was associated with increased transcript, whereas the latter was associated with reduced matrix metalloproteinase 9 activity. Rescue of muscle IR by genetic muscle-specific mitochondria-targeted catalase overexpression or by the phosphodiesterase 5a inhibitor, sildenafil, reversed HF feeding effects on ECM remodeling and increased muscle vascularity. Collagen remained elevated in HF-fed itga2(-/-) mice. Nevertheless, muscle insulin action and vascularity were increased. Muscle IR in HF-fed itga1(-/-) mice was unchanged. Insulin sensitivity in chow-fed itga1(-/-) and itga2(-/-) mice was not different from wild-type littermates.
ECM collagen expansion is tightly associated with muscle IR. Studies with itga2(-/-) mice provide mechanistic insight for this association by showing that the link between muscle IR and increased collagen can be uncoupled by the absence of collagen-integrin α(2)β(1) interaction.

Full text

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Diet-Induced Muscle Insulin Resistance Is Associated
With Extracellular Matrix Remodeling and Interaction
With Integrin a2b1in Mice
Li Kang,1Julio E. Ayala,1,2Robert S. Lee-Young,1Zhonghua Zhang,3Freyja D. James,1
P. Darrell Neufer,4Ambra Pozzi,5Mary M. Zutter,3and David H. Wasserman1,2
OBJECTIVE—The hypothesis that high-fat (HF) feeding causes
skeletal muscle extracellular matrix (ECM) remodeling in C57BL/6J
mice and that this remodeling contributes to diet-induced muscle
insulin resistance (IR) through the collagen receptor integrin a2b1
was tested.
RESEARCH DESIGN AND METHODS—The association be-
tween IR and ECM remodeling was studied in mice fed chow or
HF diet. Specific genetic and pharmacological murine models
were used to study effects of HF feeding on ECM in the absence
of IR. The role of ECM-integrin interaction in IR was studied
using hyperinsulinemic-euglycemic clamps on integrin a2b1-null
(itga22/2), integrin a1b1-null (itga12/2), and wild-type littermate
mice fed chow or HF. Integrin a2b1and integrin a1b1signaling
pathways have opposing actions.
RESULTS—HF-fed mice had IR and increased muscle collagen
(Col) III and ColIV protein; the former was associated with
increased transcript, whereas the latter was associated with
reduced matrix metalloproteinase 9 activity. Rescue of muscle IR
by genetic muscle-specific mitochondria-targeted catalase over-
expression or by the phosphodiesterase 5a inhibitor, sildenafil,
reversed HF feeding effects on ECM remodeling and increased
muscle vascularity. Collagen remained elevated in HF-fed itga22/2
mice. Nevertheless, muscle insulin action and vascularity were
increased. Muscle IR in HF-fed itga12/2mice was unchanged.
Insulin sensitivity in chow-fed itga12/2and itga22/2mice was
not different from wild-type littermates.
CONCLUSIONS—ECM collagen expansion is tightly associated
with muscle IR. Studies with itga22/2mice provide mechanistic in-
sight for this association by showing that the link between muscle
IR and increased collagen can be uncoupled by the absence of
collagen-integrin a2b1interaction. Diabetes 60:416–426, 2011
T
accompany insulin resistance in muscle are of great
he inflammatory response associated with in-
sulin resistance may have extensive effects on
extracellular matrix (ECM) remodeling and en-
dothelial cell function. ECM adaptations that
potential significance. Collagens, the most abundant struc-
tural components of ECM, not only support tissues but are
also required for cell adhesion and migration during
growth, differentiation, morphogenesis, and wound heal-
ing (1). Over 90% of the collagens expressed in skeletal
muscle are composed of collagen (Col) I, III, and IV. Al-
though ColI and ColIII comprise the fibrillar-type collagen
(1), ColIV is the most abundant structural component of the
basement membrane (2,3). Seminal studies have shown that
insulin resistant human skeletal muscle is characterized by
increased collagen (4,5). However, the functional connec-
tion between insulin resistance and ECM expansion has
received little attention. Moreover, the mechanism(s) of
changes in collagens with insulin resistance is poorly de-
fined.
Integrins are cell surface receptors that interact with
ECM and mediate both inside-out and outside-in signaling
(6,7). Of the 24 integrins identified in mammals, only four
of them bind collagens. These four collagen receptor
integrins share the b1-subunit, which heterodimerizes with
either the a1-, a2-, a10-, or a11-subunit (8). Previous studies
showed that activation of focal adhesion kinase, which is
downstream of integrin activation, was decreased in in-
sulin resistant skeletal muscle of high fat (HF)-fed rats (9).
Moreover, mice lacking integrin b1expression specifically
in striated muscle develop insulin resistance (10). These
data suggest a role for collagen-integrin interaction in the
development of insulin resistance.
The two major collagen binding receptors, integrins
a1b1and a2b1, are structurally similar but activate distinct
signaling pathways (11,12). Integrin a1b1 is antifibrotic
since integrin a1-null mesangial cells show increased re-
active oxygen species (ROS) production and collagen
synthesis (13,14). In contrast, integrin a2b1is profibrotic
since activation of integrin a2b1leads to increased ROS
production (15) and collagen expression (16,17). Integrins
a1b1and a2b1are both expressed on endothelial cells but
have opposing effects on endothelial cell biology. Integrin
a1b1is proangiogenic, and its deletion leads to decreased
endothelial cell proliferation and angiogenesis in vivo
(18,19). In contrast, integrin a2b1is antiangiogenic and its
deletion results in increased endothelial cell proliferation
and angiogenesis in vivo (20).
Herein we tested the hypothesis that the inflammatory
response associated with HF diet-induced insulin resis-
tance increases collagen in skeletal muscle of the mouse
and that this increase contributes to insulin resistance by
collagen receptor integrins a1b1 and a2b1. The specific
goals were to determine in HF-fed C57BL/6J mice whether
1) collagen accumulation in muscle occurs and the
mechanisms for any accumulation; 2) a pharmacological
or genetic manipulation that corrects muscle insulin
From the1Department of Molecular Physiology and Biophysics, Vanderbilt
University, Nashville, Tennessee; the2Mouse Metabolic Phenotyping Center,
Vanderbilt University, Nashville, Tennessee; the3Department of Pathology,
Vanderbilt University, Nashville, Tennessee; the4Department of Exercise
and Sport Science & Physiology, East Carolina University, Greenville, North
Carolina; and the5Nephrology Division, Vanderbilt University, Nashville,
Tennessee.
Corresponding author: Li Kang, li.kang@vanderbilt.edu.
Received 6 August 2010 and accepted 10 November 2010.
DOI: 10.2337/db10-1116
This article contains Supplementary Data online at http://diabetes.
diabetesjournals.org/lookup/suppl/doi:10.2337/db10-1116/-/DC1.
? 2011 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 profit,
and the work is not altered. See http://creativecommons.org/licenses/by
-nc-nd/3.0/ for details.
416 DIABETES, VOL. 60, FEBRUARY 2011diabetes.diabetesjournals.org
ORIGINAL ARTICLE

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resistance of HF feeding eliminates collagen accumulation
and increases muscle vascularization; and 3) a genetic
deletion of integrin a2b1protects against insulin resistance
and increases muscle vascularization, and a deletion of
integrin a1b1, which has actions that oppose integrin a2b1,
exacerbates insulin resistance.
RESEARCH DESIGN AND METHODS
Murine models. Mice were housed under temperature- and humidity-
controlled environment with a 12-h light/dark cycle. Distinctive murine models
were used to address specific goals of the study: 1) the association between
insulin resistance and ECM adaptations was studied in sex-matched C57BL/6J
mice fed chow or HF diet (F3282, BioServ) containing 60% calories as fat for
20 weeks; 2) novel genetic (muscle-specific mitochondrial-targeted catalase
transgenic, mcatTg) and pharmacological (sildenafil) models were used to al-
leviate insulin resistance in the presence of HF feeding, and the rescue of the
ECM adaptations was assessed; and 3) integrin a1b1-null (itga12/2) and a2b1-
null (itga22/2) mice were used to study the role of ECM-integrin interaction in
insulin resistance.
We previously showed that mcatTgmice maintained on a HF diet for 16
weeks were protected from insulin resistance (21). HF-fed mice treated with
subcutaneous injection of sildenafil, a selective phosphodiesterase (PDE)5a
inhibitor, for 13 weeks are also protected from insulin resistance (22). The
mcatTgand sildenafil-treated mice were selected as models that prevent diet-
induced muscle insulin resistance based on two considerations. First, these
approaches protect from diet-induced insulin resistance through specific
actions that target distinct mechanisms. The mcatTgmice selectively over-
express catalase in muscle mitochondria. It is a genetically modified animal
model in which the rescue of diet-induced muscle insulin resistance is related
to a reduction of mitochondrial release of ROS. This contrasts with the PDE5a
inhibition model, which causes smooth muscle relaxation and vasodilation
pharmacologically. The second consideration was that ROS have been impli-
cated in the regulation of collagen synthesis (13,23,24), and they do so by
increasing the inflammatory response and activation of mitogen-activated
protein kinase (MAPK) (25,26). Therefore, mcatTgmice provide a model for
studying the hypothesis that the inflammatory response associated with in-
sulin resistance increases collagen in muscle.
To further study the role of ECM-integrin interaction in insulin resistance,
mice lacking the two major collagen binding receptor integrins a1b1or a2b1
were studied. Male itga22/2mice and wild-type (WT) littermates (itga2+/+)
were chow-fed until 9 weeks of age at which time mice were continued on
chow or switched to HF diet until 23 weeks of age. Male itga12/2mice and WT
littermates (itga1+/+) were chow-fed until 12 weeks of age at which time mice
were continued on chow or switched to HF diet until 28 weeks of age. Murine
body composition was determined by nuclear magnetic resonance. The Van-
derbilt Animal Care and Use Committee approved animal procedures.
Hyperinsulinemic-euglycemic clamp (insulin clamp). Catheters were
implanted in a carotid artery and a jugular vein of mice for sampling and
infusions 5 days before study (27). Insulin clamps were performed on 5-h
fasted mice (28). [3-3H]glucose was primed (2.4 mCi) and continuously infused
for a 90-min equilibration period (0.04 mCi/min) and a 2-h clamp period (0.12
mCi/min). Baseline blood or plasma parameters were determined in blood
samples collected at 215 and 25 min. At t = 0, insulin infusion (4 mU/kg/min)
was started and continued for 165 min. Blood glucose was clamped at 150 ;160
mg/dL using a variable rate of glucose infusion (GIR). Mice received hepa-
rinized saline-washed erythrocytes from donors at 5 mL/min to prevent a fall of
hematocrit. Insulin clamps were validated by assessment of blood glucose
over time. Blood glucose was monitored every 10 min, and the GIR was ad-
justed as needed. Blood was taken at 80–120 min for the determination
of [3-3H]glucose. Clamp insulin was determined at t = 100 and 120 min. At
120 min, 13 mCi of 2[14C]deoxyglucose ([14C]2DG) was administered as an
intravenous bolus. Blood was taken at 2–35 min for the determination of
[14C]2DG. After the last sample, mice were anesthetized and tissues were
collected.
Insulin clamp plasma and tissue sample processing. Plasma insulin was
determined by ELISA (Millipore). Radioactivity of [3-3H]glucose, [14C]2DG, and
[14C]2DG-6-phosphate were determined by liquid scintillation counting (22).
Glucose appearance (Ra) and disappearance (Rd) rates were determined using
non-steady-state equations (29). Endogenous glucose production (endoRa)
was determined by subtracting the GIR from total Ra. The glucose metabolic
index (Rg) was calculated as previously described (30).
Immunohistochemistry. ColI, ColIII, ColIV, and Von Willebrand factor (VWF)
were assessed by immunohistochemistry in paraffin-embedded tissue sections.
Sections (5 mm) were incubated with the following primary antibodies for 60
min: anti-ColI (Abcam), anti-ColIII (CosmoBio), anti-ColIV (Abcam), or anti-VWF
(DakoCytomation). Slides were lightly counterstained with Mayer hematoxylin.
The EnVision+HRP/DAB System (DakoCytomation) was used to produce lo-
calized, visible staining. Immunostaining of CD31 was performed in either frozen
sections or paraffin-embedded sections using anti-CD31 (BD Biosciences).
Images were captured using a Q-Imaging Micropublisher camera mounted on an
Olympus upright microscope. Immunostaining was quantified by ImageJ or
BIOQUANT Life Science 2009. ColI, ColIII, and ColIV protein were measured by
the integrated intensity of staining. Muscle vascularity was determined by
counting CD31-positive structures and by measuring areas of VWF-positive
structures.
Real-time PCRs. Total RNA was isolatedfromgastrocnemius using the RNeasy
Fibrous Tissue Kit (Qiagen). Total RNA (1 mg) was reverse transcribed using the
iScript cDNA Synthesis Kit (Bio-Rad). Real-time PCR was then performed using
the SYBR Green JumpStart Taq Readymix (Sigma) on an IQ5 Multicolor Real-
Time PCR Detection System (Bio-Rad). The primers used are listed in
Supplementary Table 1. Data were analyzed by the 22DDCtmethod (31).
Gelatin zymography. Gelatin zymography was performed to measure the
activities of type IV collagenase-matrix metalloproteinase (MMP)2 and MMP9
(32). Gastrocnemius was homogenized in buffer containing 0.5% Triton X-100,
100 mM Tris-HCl, 10 mM EDTA, and 10 mL/mL protease inhibitor (pH 7.5).
Homogenates were centrifuged at 13,000 rpm for 20 min at 4°C. The super-
natant (500 mg) was incubated at 4°C for 2 h with 40 mL of gelatin-Sepharose
(Pharmacia). After the incubation, the gelatin-Sepharose beads were resus-
pended in nonreducing SDS-sample buffer and loaded on 10% zymogram gels
(Invitrogen). The gel was developed according to the manufacturer’s instruc-
tions. Conditioned medium from HT-1080 cells was used as positive control.
Western blotting. Gastrocnemius was homogenized in buffer containing
50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 1 mM EGTA, 10% glycerol, 1% Triton
X-100, 1 mM DTT, 1 mM PMSF, 5 mg/mL protease inhibitor, 50 mM NaF, and
5 mM sodium pyrophosphate and centrifuged at 13,000 rpm for 20 min at 4°C.
The supernatant (40 mg) was applied to 4–12% SDS-PAGE gel. Phosphorylated
and total Akt/protein kinase B (PKB) were probed using phospho-Akt(Ser473)
and Akt antibodies (Cell Signaling). Phosphorylated and total insulin receptor
substrate-1 (IRS-1) were probed using phospho–IRS-1 (Tyr612) and IRS-1 anti-
bodies (Upstate).
Statistical analysis. Data are expressed as means 6 SE. Statistical analyses
were performed using Student t test or two-way ANOVA followed by Turkey
post hoc tests as appropriate. The significance level was P , 0.05.
RESULTS
Diet-induced insulin resistance increases skeletal
muscle collagen expression. Twenty weeks of HF diet
in C57BL/6J mice, a regimen that induces insulin re-
sistance (33), caused inflammation in muscle, as gene ex-
pression of F4/80, a macrophage marker, and TNF-a, a
cytokine involved in systemic inflammation, was increased
in muscle of HF-fed mice compared with chow-fed mice
(Fig. 2A and B). To test the hypothesis that the inflam-
matory response associated with diet-induced insulin re-
sistance in mice is accompanied by increased muscle
collagen, we measured collagen expression in HF-fed mu-
rine muscle. Muscle ColIII and ColIV protein were increased
in HF-fed mice compared with chow-fed mice (Fig. 1A), but
protein expression of ColI was very low and differences
were undetectable.
Increases in collagen could be because of either increased
synthesis or decreased degradation via MMPs. mRNA of
specific collagen chains were used to assess collagen syn-
thesis. HF feeding increased muscle mRNA of ColIa1,
ColIa2, and ColIIIa1 chains, whereas mRNA of ColIV was
unchanged (Fig. 1B). To test whether increased ColIV pro-
tein was due to reduced degradation, the activities of type IV
collagenases, MMP2 and MMP9, were measured. HF feeding
decreased MMP9 activity, without affecting pro-MMP2 and
MMP2 activities in muscle (Fig. 1C).
Genetic and pharmacological rescue of skeletal
muscle insulin resistance after HF feeding decreases
muscle inflammation and collagen expression and
improves muscle vascularization. Muscle insulin re-
sistance was absent in HF-fed mcatTg(21) and sildenafil-
treated mice (22). In line with decreased muscle insulin
L. KANG AND ASSOCIATES
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resistance, both mcatTgand sildenafil-treated mice had
reduced F4/80 expression in muscle (Fig. 2A and C). HF
diet-induced increase in muscle TNF-a mRNA was also
eliminated in mcatTgmice (Fig. 2B); although, the decrease
in TNF-a mRNA in muscle of sildenafil-treated mice was
nearly 50%, the measurements in WT were variable and
differences were not significant (Fig. 2D). ColIII and
ColIV proteins were decreased in mcatTgand sildenafil-
treated mice (Fig. 2E). The lower expression of ColIII
in HF-fed mcatTgmice was because of reduced synthesis,
as the HF-induced increases in mRNA of ColIIIa1 were
abolished (Fig. 3B). HF-induced increase in mRNA of
ColIa2 was also abolished in mcatTgmice (Fig. 3A). De-
creased ColIV protein was due at least in part to increased
degradation via MMP9 as MMP9 activity was increased
in muscle of HF-fed mcatTgand sildenafil-treated mice
(Fig. 3C).
Decreased collagen deposition in HF-fed mcatTgand
sildenafil-treated mice was associated with improved
muscle vascularization, since blood vessel staining by
CD31 and VWF was increased (Fig. 3D). Taken together,
preventing diet-induced muscle insulin resistance using
different approaches that focus on distinct targets results
in common ECM adaptations. These common adaptations
FIG. 1. HF feeding to C57BL/6J mice increased muscle collagen expression. A: Immunohistochemical detection of ColIII and ColIV proteins in
gastrocnemius of chow-fed and HF-fed C57BL/6J mice. The magnification of images was 203. B: mRNA levels of ColIa1, ColIa2, ColIIIa1, and
ColIVa1. C: Gelatin zymogram of protein extracts from gastrocnemius of mice after either chow or HF feeding for 20 weeks. The white bands
indicate the presence of type IV collagenase activity, MMP-9, pro-MMP2, and MMP2. Data are represented as means 6 SE; n = 4 ;7. *P < 0.05 chow
vs. HF. (A high-quality color representation of this figure is available in the online issue.)
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include reduced collagen, increased MMP9 activity, and
increased vascularization.
Genetic deletion of integrin a2b1diminishes skeletal
muscle insulin resistance associated with HF feeding.
Integrins a1b1and a2b1are collagen receptors expressed
on the endothelial cell surface (19,20), as well as on sev-
eral other cell types (8). To test the role of ECM-integrin
signaling in the pathogenesis of insulin resistance, we
assessed insulin action in chow and HF-fed itga12/2and
itga22/2mice using insulin clamps. HF feeding increased
body weight, body fat, and basal 5-h fasting glucose and
insulin levels in all genotypes (Table 1). Body weights and
body fat were not different between null mice and their
respective WT littermates either on chow or HF diet. Basal
5-h fasting blood glucose did not differ between itga1+/+
and itga12/2mice regardless of diet; however, the basal
5-h fasting blood glucose was lower in the HF-fed itga22/2
mice compared with HF-fed itga2+/+mice. Blood glucose
was maintained at 150 ;160 mg/dL during the insulin
clamp in all groups (Table 1 and Fig. 4A, D, G, and J).
Plasma insulin was not different between null mice and
their respective WT littermates either in basal or insulin-
clamped state within a specific diet (Table 1).
In chow-fed mice, GIR during the insulin clamp were not
different between itga12/2and itga1+/+mice or itga22/2
and itga2+/+mice (Fig. 4B and E). Rgin muscle was also not
affected by genotype (Fig. 4C and F). HF feeding induced
insulin resistance in all groups as shown that GIR and Rg
were dramatically decreased by HF feeding (Fig. 4B, C, E,
and F vs. 4H, I, K, and L). In contrast with chow-fed mice,
GIR was lower in the HF-fed itga12/2mice compared with
HF-fed itga1+/+mice (Fig. 4H) but higher in the HF-fed
itga22/2mice when compared with HF-fed itga2+/+mice
(Fig. 4K). Muscle Rg was not different between HF-fed
itga12/2and itga1+/+mice (Fig. 4I). However, muscle Rgwas
three- to fourfold higher in HF-fed itga22/2compared with
HF-fed itga2+/+(Fig. 4L).
Because integrins a1b1and a2b1are expressed in a cell
type that is widely distributed (i.e., endothelial cells), the
metabolic phenotype of HF-fed null mice is not necessarily
FIG. 2. Rescue of muscle inflammation and collagen expansion by catalase overexpression and chronic treatment with sildenafil. A–D: Muscle
inflammation was assessed by measuring the mRNA levels of F4/80 and TNF-a using the quantitative real-time PCR. E: Immunohistochemical
detection of ColIII and ColIV proteins in gastrocnemius of mice. The magnification of images was 203. Values represent means 6 SE of integrated
intensity of staining for ColIII and ColIV. Data are normalized to WT HF or vehicle HF, and n is equal to 4 for the WT and mcatTgmice, 6 ;8 for the
vehicle- and sildenafil-treated mice. #P < 0.05 compared with WT chow. *P < 0.05 compared with WT HF or vehicle HF. (A high-quality color
representation of this figure is available in the online issue.)
L. KANG AND ASSOCIATES
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restricted to muscle. Therefore, we measured insulin ac-
tion in liver and adipose tissue, two other insulin-sensitive
glucoregulatory organs, in HF-fed integrin null mice and
WT littermates. Basal endoRawas not different between
HF-fed itga12/2and itga1+/+mice (Fig. 5A). Insulin in-
fusion decreased endoRaby 50% in the HF-fed itga1+/+mice.
In contrast, endoRawas not suppressed in the itga12/2
mice. These data are consistent with the lower GIR and
reflects greater hepatic insulin resistance in HF-fed itga12/2
mice. Rdwas not different between the HF-fed itga12/2
and itga1+/+mice either in basal or insulin-clamped state
(Fig. 5B). Insulin-stimulated adipose tissue Rg was de-
creased in HF-fed itga12/2mice relative to itga1+/+mice
(Fig. 5C). Rdwas not reduced since white adipose tissue is
a small contributor to total glucose disposal. EndoRawas
not different between the itga22/2mice and the itga2+/+mice
either in basal or insulin-clamped state (Fig. 5D); however,
Rdduring the insulin clamp was higher in itga22/2com-
pared with itga2+/+(Fig. 5E). This is consistent with in-
creased Rgin muscles. Adipose tissue Rgdid not differ
between HF-fed itga22/2and itga2+/+mice (Fig. 5F).
These data suggest that the HF-fed itga12/2mice have
a further impairment in hepatic and adipose insulin re-
sistance with no impairment in muscle insulin resistance.
HF-fed itga22/2mice have improved insulin sensitivity
because of decreased muscle insulin resistance.
Changes in muscle insulin signaling after the clamp were
consistent with flux measurements observed during the
clamp in HF-fed itga22/2mice. Phosphorylation of IRS-1
and Akt were increased in muscle of HF-fed itga22/2mice
compared with those of the itga2+/+littermates, indicative
of improved insulin signaling (Fig. 6A and B). There was
no effect on total Akt and IRS-1 in HF-fed itga22/2mice.
Decreased skeletal muscle insulin resistance in HF-fed
itga22/2mice was associated with unchanged collagen
expression and increased muscle vascularization. In
contrast with our findings in mcatTgand sildenafil-treated
mice, the HF-fed itga22/2mice were protected against
HF-induced muscle insulin resistance despite no re-
duction in muscle ColIII and ColIV proteins (Fig. 6C).
FIG. 3. Catalase overexpression and chronic treatment with sildenafil rescued muscle ECM adaptation and improved muscle vascularization. A and
B: mRNA levels of ColIa2 and ColIIIa1 in gastrocnemius of WT and mcatTgmice. C: Gelatin zymogram of protein extracts from gastrocnemius of
mice. MMP2 was not detectable in muscle under current experimental conditions. Arbitrary density of the MMP9 bands was listed. D: Immuno-
histochemical detection of vascular markers, CD31 and VWF. The magnification of images was 203. Representative images are presented. Values
represent means 6 SE of numbers of CD31-positive structures or of areas occupied by VWF-positive structures. Data are normalized to WT HF or
vehicle HF, and n is equal to 4 for the WT and mcatTgmice, 6 ;8 for the vehicle- and sildenafil-treated mice. #P < 0.05 compared with WT chow.
*P < 0.05 compared with WT HF or vehicle HF. (A high-quality digital representation of this figure is available in the online issue.)
INSULIN RESISTANCE AND COLLAGEN-INTEGRIN SIGNAL
420DIABETES, VOL. 60, FEBRUARY 2011diabetes.diabetesjournals.org