Physical activity maintains aortic endothelium-dependent

relaxation in the obese type 2 diabetic OLETF rat

Aaron K. Bunker, Arturo A. Arce-Esquivel, R. Scott Rector, Frank W. Booth, Jamal A.
Ibdah and M. Harold Laughlin
Am J Physiol Heart Circ Physiol 298:H1889-H1901, 2010. First published 19 March 2010;
doi: 10.1152/ajpheart.01252.2009
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Am J Physiol Heart Circ Physiol 298: H1889H1901, 2010.

First published March 19, 2010; doi:10.1152/ajpheart.01252.2009.

Physical activity maintains aortic endothelium-dependent relaxation in the

obese type 2 diabetic OLETF rat
Aaron K. Bunker,4 Arturo A. Arce-Esquivel,4 R. Scott Rector,1,2 Frank W. Booth,3,4,5 Jamal A. Ibdah,1,2,3
and M. Harold Laughlin3,4,5
1

Division of Gastroenterology and Hepatology, 2Harry S. Truman Memorial Veterans Medical Center, Departments
of 3Medical Pharmacology and Physiology and 4Biomedical Sciences, and 5Dalton Cardiovascular Research Center,
University of Missouri, Columbia, Missouri
Submitted 31 December 2009; accepted in final form 16 March 2010

superoxide dismutase; phosphorylated endothelial nitric oxide synthase; Otsuka Long-Evans Tokushima fatty rat
THE INCIDENCE OF type 2 diabetes mellitus (T2DM) is rising in
parallel with the presently expanding worldwide obesity epidemic. According to the American Heart Association and
Centers for Disease Control and Prevention, the diabetic and
obese population in the United States has more than doubled in
the last 15 yr (2, 3, 6). Macro- and microangiopathy are the
putative primary causes of morbidity and mortality in subjects
with T2DM, and loss of the regulatory role of the vascular

Address for reprint requests and other correspondence: M. H. Laughlin, Dept. of

Biomedical Sciences, Univ. of Missouri, E102 Veterinary Medicine Bldg., 1600 E.
Rollins Rd., Columbia, MO 65211 (e-mail: LaughlinM@missouri.edu).
http://www.ajpheart.org

endothelium is believed to be an initiating factor in the development of T2DM vascular disease (10). It is well documented
that endothelial cell dysfunction occurs in T2DM, which is
generally defined as a decrease in release of relaxing factors
and/or an increase in release of constricting factors from the
endothelium (for reviews, see Refs. 7, 16, and 46).
The Otsuka Long-Evans Tokushima fatty (OLETF) rat is a
model of T2DM and obesity selectively bred to maintain a
spontaneous mutation of the cholecystokinin-1 receptor, resulting in a within-meal feedback defect for satiety that consequently makes them hyperphagic. OLETF rats are known to
spontaneously develop obesity, insulin resistance, hyperinsulinemia, hyperglycemia, dyslipidemia, and moderate hypertension (28, 29), all of which are also major risk factors for
metabolic syndrome (15, 61) and could lead to the development of T2DM. OLETF rats are known to exhibit impaired
ACh-induced endothelium-dependent relaxation (EDR) relative to their nonmutated controls [Long-Evans Tokushima
Otsuka (LETO) rats] in the aorta (22, 25) and renal (25),
mesenteric (33, 34), and basilar arteries (35). Previous OLETF
vascular studies have reported improved ACh-induced EDR
(31) and decreased superoxide formation (30) after aortic
incubation with SOD1, one of the three SOD isoforms that
scavenges superoxide (12). ACh-induced EDR in the aorta has
also been shown to improve after incubation with a SOD1
mimetic in OLETF rats (35); however, SOD protein isoform
expression in the vasculature of OLETF rat has not been
examined. Impaired ACh-induced EDR in the OLETF rat aorta
is thought to be independent of basal nitric oxide (NO) release
and endothelial NO synthase (eNOS) protein expression as
neither have been observed to differ between diabetic OLETF
and nondiabetic control LETO rat aortas (31, 43). However,
Matsumoto et al. (36) recently showed in mesenteric arteries
that the Ser1177-phosphorylated eNOS (p-eNOS)-to-eNOS ratio (p-eNOS/eNOS ratio) was lower in OLETF rats than in
LETO rats at 60 65 wk of age. Additionally, ACh-stimulated
phosphorylation of eNOS at Ser1177 is significantly compromised and contributes in part to endothelial dysfunction in
rodent models of T2DM (51, 59). However, it is not known
whether there are temporal alterations in the OLETF aortic
p-eNOS/eNOS ratio during T2DM progression.
The importance of a physically active lifestyle for maintaining endothelial health and the prevention of cardiovascular
disease that accompanies T2DM is becoming established in
humans (for reviews, see Refs. 5, 40, and 42). Physical activity
has been shown to improve ACh-induced EDR in the thoracic
aorta and mesenteric arteries from physically active OLETF
rats compared with sedentary OLETF rats (43, 49), but obserH1889

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Bunker AK, Arce-Esquivel AA, Rector RS, Booth FW, Ibdah

JA, Laughlin MH. Physical activity maintains aortic endotheliumdependent relaxation in the obese type 2 diabetic OLETF rat. Am J
Physiol Heart Circ Physiol 298: H1889 H1901, 2010. First published
March 19, 2010; doi:10.1152/ajpheart.01252.2009.We tested the
hypothesis that physical activity can attenuate the temporal decline of
ACh-induced endothelium-dependent relaxation during type 2 diabetes mellitus progression in the Otsuka Long-Evans Tokushima fatty
(OLETF) rat. Sedentary OLETF rats exhibited decreased ACh-induced abdominal aortic endothelium-dependent relaxation from 13 to
20 wk of age (20 35%) and from 13 to 40 wk of age (3550%).
ACh-induced endothelium-dependent relaxation was maintained in
the physically active OLETF group and control sedentary Long-Evans
Tokushima Otsuka (LETO) group from 13 to 40 wk of age. Aortic
pretreatment with NG-nitro-L-arginine (L-NNA), indomethacin (Indo),
and L-NNA ! Indo did not alter the temporal decline in ACh-induced
endothelium-dependent relaxation. Temporal changes in the protein
expression of SOD isoforms in the aortic endothelium or smooth
muscle did not contribute to the temporal decline in ACh-induced
endothelium-dependent relaxation in sedentary OLETF rats. A significant increase in the 40-wk-old sedentary LETO and physically active
OLETF rat aortic phosphorylated endothelial nitric oxide (p-eNOS)to-eNOS ratio was observed versus 13- and 20-wk-old rats in each
group that was not seen in the 40- versus 13- and 20-wk-old sedentary
OLETF rats. These results suggest that temporal changes in the
antioxidant system, EDHF, and cycloxygenase metabolite production
in sedentary OLETF rat aortas do not contribute to the temporal
decline in sedentary OLETF rat aortic ACh-induced endotheliumdependent relaxation seen with type 2 diabetes mellitus progression.
We also report that physical activity in conjunction with aging in the
OLETF rat results in a temporal increase in the aortic endothelial
p-eNOS-to-eNOS ratio that was not seen in sedentary OLETF rats.
These results suggest that the sustained aortic ACh-induced endothelium-dependent relaxation in aged physically active OLETF rats may
be the result of an increase in active aortic eNOS.

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METHODS

Animals and experimental design. The animal protocol was approved by the Institutional Animal Care and Use Committee of the
University of Missouri. OLETF and LETO male rats at 4 wk of age
were kindly supplied by the Tokushima Research Institute, Otsuka
Pharmaceutical (Tokushima, Japan). Upon arrival, male OLETF and
LETO rats were immediately assigned to either physically active or
sedentary groups to be killed at 13, 20, or 40 wk of age. Nondiabetic
LETO rats with functional cholecystokinin-1 receptor gene expression
were used as controls for OLETF rats. Physically active OLETF
groups were cage confined with access to voluntary running wheels
equipped with a Sigma Sport BC 606 bicycle computer (Cherry Creek
Cyclery, Foster Falls, VA), and sedentary groups were cage confined
without running wheel access. Voluntary wheel running was selected
to simulate the more natural activity state and lifestyle of the animal.
All rats had ad libitum access to water and rat chow (Formulab 5008,
Purina Mills, St Louis, MO) and were housed 1 rat/cage in a room
with a 06.00 18.00 h light:18.00 06.00 h dark cycle at 20 22C,
which was maintained throughout the experimental period.
At 13, 20, and 40 wk of age (n " 5 8 rats/group), rats were killed
with running wheels locked 53 h (53WL) before death. Based on
previous unpublished data from this laboratory, 53WL was not expected to alter the beneficial long-term effects of regular physical
activity on aortic vasomotor function as the beneficial effects of
physical activity on endothelial function have been shown to last up to
2 days after the cessation of regular physical activity in rats (18).
Additionally, 0-h postphysical activity reveals a transient decrease in
endothelial function (18), further supporting the rationale for the time
point of 53WL. 53WL also permitted us to examine the effects of a
regular physically active lifestyle and not the acute effects after a
AJP-Heart Circ Physiol VOL

recent bout of physical activity. Additionally, rats were fasted 5 h

before death for accurate serum measurements.
Rats were deeply anesthetized with pentobarbital sodium (100
mg/kg) for tissue removal and terminated via heart exsanguination.
Before heart exsanguination, blood was collected using a hypodermic
syringe for the determination of plasma glucose and insulin levels to
evaluate the maintenance of glycemic control in all rats at each
experimental time point. Rat body weights were measured before
anesthesia, and wet weights of the hearts were taken after exsanguinations using a standard laboratory balance. Heart weight-to-body
weight ratios were determined as a measure of the extent of physical
activity as well as citrate synthase activity (CSA) in the red portion of
the gastrocnemius muscle (methods described below).
Dual-energy X-ray absorptiometry. Whole body composition was
measured using a Hologic QDR-1000/w dual-energy X-ray absorptiometry machine calibrated for rats while rats were under anesthesia,
as performed previously at this institution (32).
Assessment of vascular function. The vessel functional experimental protocols described here and some drug concentrations are similar
to previous experiments conducted in this laboratory (23). To evaluate
changes in the temporal profile of vascular function during T2DM
disease progression in the OLETF rat, at 13, 20, and 40 wk of age the
abdominal aortas were dissected and cleaned of connective and
adipose tissue after rats had been killed via heart exsanguination. The
abdominal aorta was then cut into 3-to 4-mm-long vessel rings with
the first ring being cut at the distal abdominal aorta; from there, the
remaining rings were cut moving proximal up the vessel. Cut rings
were photographed on an Olympus video microscope for ring morphological characteristic measurements using Image-J software. Rings
were then mounted on wire feet connected to isometric force transducers and submerged in 20-ml water baths containing physiological
Krebs solution maintained at 37C for 1 h to allow for equilibration.
Aortic rings were stretched to a length that produced maximal force
stimulated by 60 mM KCl. Once that length was set, two separate
maximal constrictions induced by 80 mM KCl were conducted on all
rings to analyze vessel contractility. Aortic vasomotor function was
investigated with cumulative concentration-response curves of vasoactive agents conducted in the following order: ACh (half logconcentration increments ranging from 1 e#10 to 1 e#4 M) and sodium
nitroprusside (SNP; whole log-concentration increments ranging from
1 e#10 to 1 e#4 M). A submaximal concentration of phenylephrine
(3 e#7 M) was used to preconstrict all vessels before ACh and SNP
relaxation curves. We used a pharmacological approach to examine
the contribution of different endothelial signaling pathways in EDR.
We used NG-nitro-L-arginine (L-NNA) to inhibit eNOS, the enzyme
that produces NO. We used indomethacin (Indo) to inhibit cyclooxygenase (COX), the enzyme responsible for prostacyclin and prostaglandin production. We used combined treatment with L-NNA and
Indo to block both COX and NOS, leaving only the non-NOS,
non-COX pathways to induce EDR (likely EDHF). All cumulative
concentration-response curves were conducted on four aortic rings
that were pretreated as follows: control/untreated (in Krebs solutions
only), 20-min preincubation with L-NNA (3 e#4 M in Krebs solution),
20-min preincubation with Indo (5 e#6 M in Krebs solution), and
20-min preincubation with both Indo and L-NNA (in Krebs solution).
Immunoblots. After exsanguination, the thoracic aorta was dissected and stripped of connective and adipose tissue. The aorta was
opened from end to end using a scalpel, and endothelial cells were
scraped off into ice-cold Laemmli buffer and stored at 80C for
SOD1, SOD2, SOD3, eNOS, and Ser1177 p-eNOS immunoblot analysis. Segments of the thoracic aorta that had been scraped free of
endothelial cells were also stored in Laemmli buffer at #80C for future
vascular smooth muscle cell SOD1, SOD2, eNOS, and p-eNOS immunoblot analysis. Immunoblot analysis for protein expression in vascular
smooth muscle and endothelial cells was done following methods used
previously in this laboratory (39). Briefly, a NanoOrange protein assay
was used to determine the sample protein content (in $g/$l) using a
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vations were not made throughout the progression of T2DM.

Little is also known about whether physical activity maintains
the vascular SOD antioxidant system and the p-eNOS/eNOS
ratio compared with a sedentary lifestyle during the progression of T2DM. Studies in aortas from diabetic and nondiabetic
rodent models have shown increases in SOD isoform protein
expression in response to exercise (9, 44), and regular physical
activity/exercise is also known to be a powerful stimulus for
the phosphorylation of eNOS in humans (17) and rodents (19,
60). A study (36) on OLETF rats has shown that the mesenteric
artery p-eNOS/eNOS ratio is lower in OLETF rats versus
LETO rats. However, in all these studies observations were
made only at a single age and not throughout the progression
of T2DM. In addition, studies using the OLETF rat, a T2DM
rat model, to examine whether physical activity alone (i.e.,
voluntary running on running wheels) maintains the vascular
SOD antioxidant system and p-eNOS/eNOS ratio during the
progression of T2DM are nonexistent.
The primary aim of this study was to evaluate whether a
primary preventative therapy such as regular physical activity
can attenuate the temporal decline of ACh-induced EDR during the progression of T2DM. Our hypothesis was that a
sedentary lifestyle contributes to the reduction in ACh-induced
EDR in the sedentary OLETF aorta associated with T2DM
disease progression due to a decreased bioavailability of NO
resulting from the downregulated expression of endothelial
SOD isoform(s) and a reduction in the p-eNOS/eNOS ratio. It
was also hypothesized that physical activity would maintain
ACh-induced EDR in part by temporally preserving endothelial SOD isoform(s) protein expression and the p-eNOS/eNOS
ratio in the OLETF rat aorta.

AORTIC FUNCTION: TYPE 2 DIABETES AND PHYSICAL ACTIVITY

Scientific. All equipment used for gels and immunoblot transfer was
purchased from Invitrogen. Polyclonal antibodies for SOD1, SOD2,
and SOD3 were purchased from Stressgen, and eNOS and p-eNOS
were purchased from BD Transduction. Anti-rabbit and anti-mouse
secondary antibodies were purchased from Sigma and GE Healthcare,
respectively. SuperSignal visualization reagent was purchased from
Pierce ThermoScientific.
Statistics. Differences between groups regarding immunoblot data,
logEC50 values, serum and plasma measurements, heart weight, body
weight, heart weight-to-body weight ratio, percent body fat, CSA,
food consumption, and ring characteristics were determined via oneway ANOVA using GraphPad Prism version 5.0a. Significant main
effects (P % 0.05) were followed up with Fisher least-significantdifference post hoc comparisons.
The analysis of concentration-response curves was performed using the Mixed procedure in SAS version 9 to allow for heterogeneous
variances across doses. The statistical model used was a two-factor
study with age being a between-subjects factor and drug concentration
being a within-subjects factor. Residual plots were examined to check
on the assumption of normally distributed error terms. There were
some instances of convergence problems when running the mixed
model for certain combinations of group and treatment. In these cases,
a series of nonparametric analyses was run to look for differences in
age groups. Specifically, the Kruskal-Wallis test was used at each
concentration level separately. When results were significant, pairwise
comparisons were performed to determine which age groups significantly differed from others. Given the large number of tests that were
ran, the false discovery rate adjustment for multiple tests was used.
Differences with false discovery rate-adjusted P values of %0.05 (P %
0.05) were considered significant.
RESULTS

Animal characteristics. Animal characteristics are shown in

Table 1. Consistent with our previous reports (32, 47), the
average daily voluntary running distance in the physically
active OLETF rats was &9 km/day at 13 wk, &7 km/day at 20
wk, and &4 km/day at 40 wk (data not shown). Body masses
and body fat percentages were significantly greater in sedentary OLETF animals compared with sedentary LETO rats and
physically active OLETF rats at all ages studied. A higher heart
weight-to-body weight ratio, a marker of exercise training, was
found higher in physically active OLETF rats at all ages

Table 1. Animal characteristics

Percent Body Fat

Sedentary LETO rats

13 wk old
20 wk old
40 wk old
Sedentary OLETF
rats
13 wk old
20 wk old
40 wk old
Physically active
OLETF rats
13 wk old
20 wk old
40 wk old

Body Weight, g

Heart Weight-to-Body
Weight Ratio, mg/g

Red Gastrocnemius Muscle

Citrate Synthase Activity

Food Consumption, gwk#1g

body wt#1

10.0 ' 0.5

15.5 ' 1.1*
22.4 ' 1.3*

380.8 ' 5.9

477.9 ' 6.6*
557.4 ' 15*

3.4 ' 0.1

2.9 ' 0.1*
2.6 ' 0.1*

207.1 ' 20.6

270.8 ' 31.2*
160.6 ' 26.4

0.36 ' 0.01

0.33 ' 0.01
0.30 ' 0.01*

21.7 ' 1.8

30.4 ' 0.8*
30 ' 4.0*

495.4 ' 15.7

607.0 ' 10.5*
685.2 ' 34.6*

3.1 ' 0.1

2.7 ' 0.07*
2.7 ' 0.1*

157.9 ' 7.9

253.4 ' 20.7*
112.3 ' 10.7

0.38 ' 0.01

0.35 ' 0.01
0.47 ' 0.05*

7.4 ' 0.6

11.4 ' 1.9*
17.3 ' 1.9*

368.0 ' 10.8

434.7 ' 10.5*
544.5 ' 15.8*

4.0 ' 0.1

3.6 ' 0.1*
3.5 ' 0.1*

200.6 ' 17.3

334.7 ' 32.3*
158.5 ' 17.6

0.55 ' 0.02

0.52 ' 0.02
0.43 ' 0.01

Values are means ' SE; n " 5 8 rats/group. Values were obtained from 5-h fasted rats. LETO rats, Long-Evans Tokushima Otsuka rats; OLETF rats, Otsuka
Long-Evans Tokushima fatty rats. Significant differences between groups or ages are denoted with the following symbols: *P % 0.05 vs. 13 wk within the
respective group, P % 0.05 vs. sedentary LETO rats at the respective age, P % 0.05 vs. physically active OLETF rats at the respective age, and P % 0.05
vs. 20 wk within the respective group.
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Tecan Safire Plate reader (model A-5082). Samples containing equal

amounts of aortic proteins (10 $g) were loaded onto a 15-well 4 12%
bis-Tris gel, subjected to electrophoresis, and then transferred to a
polyvinylidene difluoride membrane. Membranes were blocked in a
5% nonfat milk-Tris-buffered saline-Tween 20 (TBST) solution and
incubated overnight with a primary antibody against SOD1 (19 kDa,
1:5,000), SOD2 (25 kDa, 1:1,000), SOD3 (35 kDa, 1:500), eNOS
(140 kDa, 1:1,000), or p-eNOS (140 kDa, 1:250). Next, membranes
were incubated with a horseradish peroxidase-conjugated anti-rabbit
or anti-mouse secondary antibody in a 5% nonfat milk-TBST solution.
For the analysis of p-eNOS expression, the detection of phosphoprotein was followed by membrane stripping in stripping buffer at 50C
for 30 min followed by the detection of total eNOS expression. Blots
for all proteins were then incubated in Pierce SuperSignal West Dura
visualization reagent for 5 min, and a Kodak image station (4000R)
was used to visualize and quantify protein band densities. All Western
blot data were normalized to 13-wk values, allowing us to compare
temporal changes in vascular protein. Data were not obtained regarding SOD3 protein expression because SOD3 antibodies did not detect
any protein. This is consistent with reports (4, 27) showing that SOD3
is virtually undetectable and/or absent in rat uterine, lung, and vascular tissue.
Citrate synthase activity. The red portion of the gastrocnemius
muscle was dissected and frozen in liquid nitrogen to be stored at
#80C for an assay of CSA as a measure of the adaptive response to
physical activity on skeletal muscle oxidative capacity, as previously
described by this laboratory (39) using the methods of Srere (50).
Serum and plasma NO metabolite measurements. Serum glucose
(Sigma), triglycerides (Sigma), and insulin (Linco Research) were
measured using commercially available kits according to the manufacturers instructions. Hemoglobin A1c (HbA1c) concentrations
were determined as previously described by our group (32). The
plasma NO metabolite (nitrite ! nitrate; NOx) concentration was
determined via VCl3-induced reduction of NOx to NO and its subsequent reaction with O3, producing chemiluminescence (model NOA
280i, Sievers) as has been previously done in this laboratory (41).
Chemicals, solutions, and drugs. The Krebs-bicarbonate buffer
solution contained (in mM) 131.5 NaCl, 5.0 KCl, 1.2 NaH2PO4, 1.2
MgCl2, 2.5 CaCl2, 11.2 glucose, 20.8 NaHCO3, 0.003 propranolol,
and 0.025 EDTA. The solution was aerated with 95% O2-5% CO2 (pH
7.4) and maintained at 37C. The stripping buffer for immunoblots
contained 62.5 mM Tris base, 2% SDS, and 100 mM 2-mercaptoethanol (pH 6.7). ACh, SNP, L-NNA, and Indo were purchased from
Sigma, and all other chemicals were purchased from Sigma or Fisher

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significantly higher plasma NOx levels than physically active

OLETF rats at 13 and 20 wk of age. Physically active OLETF
rat plasma NOx levels returned to control LETO rat levels by
40 wk. Physically active OLETF rats at 40 wk (10.3 ' 0.4
nM/ml media) had significantly higher NOx levels than sedentary OLETF rats at 40 wk (6.4 ' 0.3nM/ml media).
Vessel characteristics. No differences were found in vessel
characteristics (outer diameter, inner diameter, vessel length,
and preconstriction induced by phenylephrine; data not shown)
between rat groups at each age or between ages in each rat
group. In addition, no differences were found in specific
tension responses elicited by 80 mM KCl between ages in each
rat group or between rat groups at each age except that
13-wk-old physically active OLETF rats (0.8 ' 0.13 g/mm2)
had significantly (P % 0.05) less specific tension than 13-wkold sedentary LETO rats (1.4 ' 0.6 g/mm2).
Relaxation responses to ACh. Temporal alterations in AChinduced aortic responses of sedentary LETO rats or physically
active OLETF rats were not observed among 13-, 20-, and
40-wk-old sedentary LETO rat or 13-, 20-, and 40-wk-old
physically active OLETF rat control/untreated aortic rings
(Fig. 1, A and C). In contrast, rat aortic rings from 13 to 20 wk
in sedentary OLETF rats exhibited a significant decrease (20
25%) of the relaxation response to ACh (1 e#61 e#4 M), and
this response continued to decrease (3550%) from 13 to 40
wk (1 e#71 e#4 M) in sedentary OLETF rat aortic rings (Fig.
1E). Pretreatment with an inhibitor of eNOS, L-NNA, blocked
the aortic ring relaxation responses to ACh at each age equally
in each rat group (Fig. 1, B, D, and F). Pretreatment with Indo,
an inhibitor of the enzyme COX that is responsible for prostacyclin and prostaglandin production, revealed that 20-wk-old
sedentary LETO rat aortic rings exhibited significantly less
ACh relaxation than 13- and 40-wk-old sedentary LETO rat
aortic rings (3 e#81 e#4 M; Fig. 2A). No significant differences in Indo-pretreated aortic ring relaxation responses to
ACh were observed among 13-, 20-, and 40-wk-old physically
active OLETF rats (Fig. 2C) or 13-, 20-, and 40-wk-old
sedentary OLETF rats (Fig. 2E). Combined pretreatment of
aortic rings with L-NNA and Indo did not result in any
significant differences among 13-, 20-, and 40-wk-old sedentary LETO rats (Fig. 2B) or 13-, 20-, and 40-wk-old physically
active OLETF rats (Fig. 2D). Pretreatment of 20-wk-old sed-

Table 2. Serum and plasma measurements

Glucose, mg/dl

Sedentary LETO rats

13 wk old
20 wk old
40 wk old
Sedentary OLETF rats
13 wk old
20 wk old
40 wk old
Physically active OLETF rats
13 wk old
20 wk old
40 wk old

Hemoglobin A1c, %

Insulin, ng/ml

268.3 ' 26.1

331.2 ' 37.9
261.3 ' 20.1

4.7 ' 0.1

4.6 ' 0.1
4.6 ' 0.1

8.4 ' 1.1

9.0 ' 0.8
10.7 ' 0.7

393.5 ' 34.7

541.5 ' 57.7*
665.9 ' 41.8*

5.4 ' 0.1

5.3 ' 0.1
8.8 ' 0.7*

10.4 ' 1.2

12.5 ' 0.7
5.0 ' 1.2*

256.8 ' 26.3

380.3 ' 52.7
301.5 ' 16.6

4.8 ' 0.1

4.7 ' 0.1
4.8 ' 0.1

8.3 ' 1.4

9.6 ' 1.1
11.7 ' 0.8

Triglycerides, mg/dl

36.8 ' 4.7

42.7 ' 4.3
42.3 ' 3.1
114.7 ' 18.2
177.1 ' 24.8*
253.4 ' 32.8*
52.7 ' 5.7
71.3 ' 10.8
81.0 ' 9.7

NOx, mM/ml Media

12.8 ' 1.3

12.2 ' 1.4
10.4 ' 0.7
7.5 ' 0.8
7.3 ' 1.0
6.4 ' 0.3
5.4 ' 0.5
6.1 ' 0.8
10.3 ' 0.4*

Values are means ' SE; n " 5 8 rats/group for serum measurements and 4 rats/group for plasma nitric oxide metabolite (NOx) measurements. Values were
obtained from 5-h fasted rats. Significant differences between groups or ages are denoted with the following symbols: *P % 0.05 vs. 13 wk within the respective
group, P % 0.05 vs. sedentary LETO rats at the respective age, P % 0.05 vs. physically active OLETF rats at the respective age, and P % 0.05 vs. 20 wk
within the respective group.
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compared with sedentary LETO rats and sedentary OLETF

rats. In addition, CSA in the red gastrocnemius muscle increased in all groups from 13 to 20 wk but returned back to
13-wk levels by 40 wk and was significantly greater in physically
active OLETF rats compared with sedentary OLETF rats in only
the 20-wk-old group. Average food consumption per week relative to body weight was significantly greater in physically active
OLETF rats compared with sedentary LETO rats at each age and
significantly greater than sedentary OLETF rats at 13 and 20 wk,
suggesting that observations in the hyperphagic physically active
OLETF rats are not due to the consumption of less calories.
Serum and plasma NOx measurements. Serum and plasma
NOx measurements are shown in Table 2. Sedentary OLETF
rats displayed insulin resistance by 13 and 20 wk of age, as
shown by elevated levels of postprandial serum glucose at 13
wk and serum insulin at 20 wk compared with physically active
OLETF rats and sedentary LETO rats. By 40 wk, sedentary
OLETF animals developed overt T2DM with a 60% drop in
serum insulin and an approximately twofold increase in
HbA1c, whereas physically active OLETF rats maintained
normal glycemic control to the level of nonobese, nonhyperphagic sedentary LETO rats at all ages examined. In addition, daily voluntary wheel running maintained serum triglyceride levels in physically active OLETF rats to the level of
sedentary LETO rats, which was significantly reduced compared with sedentary OLETF rats at 13, 20, and 40 wk of age.
The serum glucose, HbA1c, insulin, and triglyceride data
collectively illustrate the importance of regular physical activity in the maintenance of glycemic control and circulating lipid
levels in these hyperphagic rats. Additionally, the percent body
fat data in conjunction with serum glucose, HbA1c, insulin,
and triglyceride data further illustrate the importance of regular
physical activity in the prevention of metabolic syndrome in
these hyperphagic rats.
Significant temporal differences in plasma NOx were only
observed in the physically active OLETF group, where NOx
was significantly higher in 40-wk-old (10.3 ' 0.4 nM/ml
media) compared with 13-wk-old (5.4 ' 0.5 nM/ml media)
and 20-wk-old (7.3 ' 1.0 nM/ml media) physically active
OLETF rats. Within-group comparisons of plasma NOx revealed that control LETO rats had significantly higher NOx
plasma levels than sedentary OLETF rats at each age and

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entary OLETF rat aortic rings with L-NNA ! Indo failed to

completely block ACh relaxation compared with aortic rings
from 13- and 40-wk-old sedentary OLETF rats (1 e#81 e#4
M; Fig. 2F), suggesting the presence of non-NOS-, non-COXinduced relaxation. In aortic rings that had been denuded,
meaning that the endothelial cell layer was scraped off with
forceps (n " 3 4), ACh-induced aortic ring relaxation responses were equally abolished at 13, 20, and 40 wk within
each rat group, suggesting that the denuded rings were essentially free of endothelial cells (data not shown). Additionally,
no differences were found among the logEC50 values at each
age within each rat group for the ACh concentration-response
curves in the presence or absence of inhibitors (data not
shown).
Relaxation responses to SNP. No significant differences in
the relaxation response to SNP were observed among 13-, 20-,
and 40-wk-old sedentary LETO rat control/untreated aortic
rings (Fig. 3A). In contrast, physically active OLETF rat
control/untreated aortic rings at 40 wk (#7.75 ' 0.14 logEC50)
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showed decreased sensitivity to SNP, as demonstrated by a

significant rightward shift in the SNP concentration-response
curve compared with those of 13-wk-old (#8.65 ' 0.15
logEC50) and 20-wk-old (#8.48 ' 0.11 logEC50) physically
active OLETF rats (Fig. 3C). Sedentary OLETF rat control/
untreated aortic rings at 40 wk (#7.60 ' 0.15 logEC50) also
demonstrated a significant rightward shift in the SNP concentration-response curve compared with those of 13-wk-old
(#8.61 ' 0.19 logEC50) and 20-wk-old (#8.37 ' 0.11
logEC50) sedentary OLETF rats (1 e#91 e#6 M; Fig. 3E).
Pretreatment with L-NNA did not affect SNP-induced relaxation responses in the 13-, 20-, and 40-wk-old LETO rat groups
(Fig. 3B). The rightward shift in SNP-induced relaxation responses persisted after pretreatment with L-NNA at 40 wk
(#8.23 ' 0.15 logEC50) compared with 13-wk (#8.88 ' 0.15
logEC50) and 20-wk (#8.90 ' 0.13 logEC50) responses in
physically active OLETF rats (Fig. 3C). Similarly, in sedentary
OLETF rats, the rightward shift in SNP-induced relaxation
responses persisted after pretreatment with L-NNA at 40 wk
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Fig. 1. Concentration-response curves for the

ACh-induced relaxation of isolated abdominal
aortic rings obtained from sedentary LongEvans Tokushima Otsuka sedentary (LETO)
rats (LSED), physically active Otsuka LongEvans Tokushima fatty (OLETF) rats (OPA),
and sedentary OLETF rats (OSED) at 13, 20,
and 40 wk of age. Data were obtained in the
absence (A, C, and E) or presence (B, D, and F)
of NG-nitro-L-arginine [L-NNA (LN); 3 e#4
M]. See METHODS for more details. Data are
means ' SE; n " 6 8 rats per group or
treatment combination. *P % 0.05, 20 vs. 13
wk; #P % 0.05, 40 vs. 13 wk; P % 0.05, 40 vs.
20 wk.

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(#7.70 ' 0.06 logEC50) compared with 13-wk (#8.82 ' 0.09
logEC50) and 20-wk (#8.49 ' 0.19 logEC50) sedentary
OLETF responses (Fig. 3D). There were no significant differences among 13-, 20-, and 40-wk sedentary LETO rat aortic
responses (Fig. 4A) or 13-, 20-, and 40-wk physically active
OLETF rat aortic responses to SNP after treatment with Indo
(Fig. 4C). These results in physically active OLETF aortas
suggest that Indo abolished the rightward shift in SNP relaxation responses in 40-wk-old physically active OLETF rat
aortic rings. Sedentary OLETF rat aortic rings pretreated with
Indo continued to exhibit a significant rightward shift in the
SNP dose-response relaxation curve at 40 wk (#8.13 ' 0.15
logEC50) compared with 13-wk-old (#9.29 ' 0.16 logEC50)
and 20-wk-old (#9.15 ' 38 logEC50) sedentary OLETF rats
(Fig. 4E). Combined pretreatment of aortic rings with L-NNA !
Indo had effects similar to the effects of Indo pretreatment
alone in control LETO rats and physically active OLETF rats
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(Fig. 4, B and D). Sedentary OLETF rat aortic responses to SNP

after combined pretreatment of aortic rings with L-NNA ! Indo
continued to result in a significant rightward shift in 40-wk-old
(#8.24 ' 0.07 logEC50) sedentary OLETF rat responses
compared with 13-wk-old (#9.10 ' 0.18 logEC50) and 20wk-old (#8.94 ' 0.14 logEC50) sedentary OLETF rat responses. In aortic rings that had been denuded (n " 3 4),
SNP-induced aortic ring relaxation responses were equally
unaffected by denudation at 13, 20, and 40 wk within each rat
group (data not shown). Unless otherwise noted, no differences
were found between logEC50 values at each age within each rat
group for the SNP concentration-response curves in the presence or absence of inhibitors (data not shown).
Aortic vascular smooth muscle cell protein expression of
SOD1 and SOD2. Immunoblot analysis revealed no significant
differences among 13-, 20-, and 40-wk thoracic aorta smooth
muscle SOD2 protein expression in any rat group (Fig. 5A).
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Fig. 2. Concentration-response curves for

the ACh-induced relaxation of isolated abdominal aortic rings obtained from LSED,
OPA, and OSED rats at 13, 20, and 40 wk of
age. Data were obtained in the presence of
indomethacin (Indo; 5 e#6 M; A, C, and E)
or combined L-NNA ! Indo (B, D, and F).
See METHODS for more details. Data are
means ' SE; n " 6 8 rats per group or
treatment combination. *P % 0.05, 20 vs. 13
wk; #P % 0.05, 20 vs. 40 wk.

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SOD1 thoracic aorta smooth muscle protein expression in

40-wk-old sedentary LETO rats was higher compared with 13and 20-wk-old sedentary LETO animals (Fig. 5B), but no
differences among 13-, 20-, and 40-wk-old sedentary OLETF
rats or 13-, 20-, and 40-wk-old physically active OLETF
thoracic aorta smooth muscle SOD1 protein levels were observed (Fig. 5B).
Aortic endothelial cell protein expression of SOD1, SOD2,
eNOS, and p-eNOS. Immunoblot analysis revealed no significant differences among 13-, 20-, and 40-wk-old thoracic aorta
endothelial SOD2 protein expression in sedentary LETO and
physically active OLETF rat groups (Fig. 6A). Thoracic aorta
endothelial SOD2 protein levels in 20-wk-old sedentary
OLETF rats were significantly lower compared with 13- and
40-wk-old sedentary OLETF rats (Fig. 6A).
Thoracic aorta endothelial SOD1 protein expression in 20wk-old sedentary LETO rats was significantly higher than in
13- and 40-wk-old sedentary LETO animals, whereas no significant differences among 13-, 20-, and 40-wk thoracic aorta
endothelial SOD1 protein expression in sedentary OLETF and
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physically active OLETF rat groups were observed (Fig. 6B).

Thoracic aorta endothelial eNOS protein expression was not
significantly different among 13-, 20-, and 40-wk-old rats
within any group (Fig. 7A). Thoracic aorta endothelial protein
levels of p-eNOS (Ser1177) at 40 wk was significantly greater
compared with 13- and 20-wk-old rats in the sedentary LETO
and physically active OLETF rat groups; however, no differences were found in sedentary OLETF rat thoracic aorta
endothelial p-eNOS (Ser1177) expression at 13, 20, and 40 wk
(Fig. 7B). Examination of the p-eNOS/eNOS ratios shown in
Fig. 8B showed that relative to the total pool of available eNOS
in the thoracic aorta endothelium, the percentage of p-eNOS at
Ser1177 was significantly greater in 40-wk-old rats compared
with 13- and 20-wk-old rats in the sedentary LETO and
physically active OLETF rat groups. Concurrent with this
temporal increase in p-eNOS at Ser1177 in 40-wk-old physically active OLETF rats was a significantly augmented plasma
NOx concentration in 40-wk-old physically active OLETF rats
compared with 13- and 20-wk-old physically active OLETF
rats (Table 2). This temporal increase in plasma NOx was not
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Fig. 3. Concentration-response curves for the

sodium nitroprusside (SNP)-induced relaxation of isolated abdominal aortic rings obtained from LSED, OPA, and OSED rats at
13, 20, and 40 wk of age. Data were obtained
in the absence (A, C, and E) or presence (B,
D, and F) of L-NNA (3 e#4 M). See METHODS
for more details. Data are means ' SE; n "
6 8 rats per group or treatment combination.
*P % 0.05, 40 vs. 13 wk; #P % 0.05, 40 vs.
20 wk.

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AORTIC FUNCTION: TYPE 2 DIABETES AND PHYSICAL ACTIVITY

observed in sedentary LETO or sedentary OLETF groups

(Table 2) and demonstrates the importance of physical activity
as a stimulus to return plasma NOx levels in OLETF rats to
levels similar to those of LETO control rats.
DISCUSSION

The purpose of this study was to evaluate whether physical activity can attenuate the temporal decline in AChinduced EDR in the OLETF rat abdominal aorta during the
progression to overt T2DM. Our hypothesis was that a
sedentary lifestyle contributes to a reduction in ACh-induced EDR in the OLETF aorta during T2DM disease
progression due to decreased bioavailability of NO resulting
from the downregulated expression of endothelial SOD
isoform(s) and a reduction in the Ser1177 p-eNOS/eNOS
ratio. We hypothesized that physical activity would maintain ACh-induced EDR in part by preserving endothelial
SOD protein content and the p-eNOS/eNOS ratio. We found
that physical activity indeed preserved ACh-induced EDR in
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the physically active OLETF abdominal aorta, whereas

ACh-induced EDR progressively decreased in sedentary
OLETF rats in association with the development of T2DM.
Contrary to our hypothesis, it does not appear that the
temporal decrease in ACh-induced EDR in sedentary
OLETF rats was due to the downregulated expression of
endothelial SOD1 and SOD2 isoforms during the progression to T2DM as there were no significant differences in the
protein content of SOD1 and SOD2 isoforms in sedentary
OLETF rats across ages. Furthermore, contrary to our hypothesis, the p-eNOS/eNOS ratio was maintained by sedentary OLETF rats during T2DM progression. However, in
LETO rats and in physically active OLETF rats, there was a
significant increase in the p-eNOS/eNOS ratio in the aortic
endothelium at 40 wk of age that was not seen in the
sedentary OLETF group at 40 wk of age. These results
illustrate the importance of physical activity in elevating the
active state of eNOS in the OLETF rat aorta during the time
of progression to the insulin-dependent stage of T2DM.
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Fig. 4. Concentration-response curves for

the SNP-induced relaxation of isolated abdominal aortic rings obtained from LSED,
OPA, and OSED rats at 13, 20, and 40 wk of
age. Data were obtained in the presence of
Indo (5 e#6 M; A, C, and E) or combined
L-NNA ! Indo (B, D, and F). See METHODS
for more details. Data are means ' SE; n "
6 8 rats per group or treatment combination. *P % 0.05, 40 vs. 13 wk; #P % 0.05, 40
vs. 20 wk.

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ACh-induced EDR, suggesting that the decline in ACh-induced EDR is a consequence of T2DM progression rather than
a contributing factor to the development of T2DM. The temporal decline in ACh-induced EDR in the sedentary OLETF
abdominal aorta and T2DM progression was prevented by
physical activity in physically active OLETF rats. These data
indicate the importance of regular physical activity in maintaining endothelial function and the prevention of T2DM and
obesity in the OLETF rat.
The temporal maintenance of aortic endothelial function in
physically active OLETF rats did not appear to be mediated via
temporal alterations in SOD2 or SOD1 expression within aortic
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Fig. 5. Analysis of SOD2 (A) and SOD1 (B) protein expression in thoracic
aorta segments scraped free of endothelial cells obtained from LSED, OSED,
and OPA rats at 13, 20, and 40 wk of age. Top: representative Western blots
for SOD2 and SOD1 in each rat group at each age. Bottom: bands were
quantified as described in METHODS. Ratios were calculated for the optical
density of each SOD isoform at 13, 20, or 40 wk of age over that of 13-wk-old
rats in each respective group. Values are means ' SE of 6 8 determinations.
*P % 0.05 vs. 13 wk in each respective group; #P % 0.05 vs. 20 wk in each
respective group.

A primary finding in the present report, as indicated by the

results shown in Fig. 1, A, C, and E, supports our hypotheses
that a sedentary lifestyle contributes to a temporal reduction in
ACh-induced EDR in the OLETF abdominal aorta during
T2DM disease progression and that physical activity acts to
temporally maintain ACh-induced EDR in the OLETF abdominal aorta during T2DM progression. The findings reported in
this study are the first to show that during the transition from
insulin resistance to overt T2DM there is a concurrent temporal
decline in ACh-induced EDR in the OLETF abdominal aorta.
Additionally, at 13 wk of age, sedentary OLETF rats were
insulin resistant (Table 2) before the observed decline in
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Fig. 6. Analysis of SOD2 (A) and SOD1 (B) protein expression in endothelial
cells scraped from segments of the thoracic aorta obtained from LSED, OSED,
and OPA rats at 13, 20, and 40 wk of age. Top: representative Western blots
for SOD2 and SOD1 in each rat group at each age. Bottom: bands were
quantified as described in METHODS. Ratios were calculated for the optical
density of each SOD isoform at 13, 20, or 40 wk of age over that of 13-wk-old
rats in each respective group. Values are means ' SE of 6 8 determinations.
*P % 0.05 vs. 13 wk in each respective group; #P % 0.05 vs. 40 wk in each
respective group.
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Fig. 7. Analysis of endothelial nitric oxide synthase (eNOS; A) and phosphorylated eNOS (p-eNOS; Ser1177; B) protein expression in endothelial cells
scraped from segments of the thoracic aorta obtained from LSED, OSED, and
OPA rats at 13, 20, and 40 wk of age. Top: representative Western blots for
eNOS and p-eNOS in each rat group at each age. Bottom: bands were
quantified as described in METHODS section. Ratios were calculated for the
optical density of eNOS or p-eNOS at 13, 20, or 40 wk over that of 13-wk-old
rats in each respective group. Values are means ' SE of 6 8 determinations.
*P % 0.05 vs. 13 wk in each respective group; #P % 0.05 vs. 20 wk in each
respective group.

smooth muscle (Fig. 5, A and B) or endothelial cells (Fig. 6, A

and B). Previous studies (9, 44) using rodents that have shown
exercise-induced increases SOD2 and SOD1 protein expression differed from ours in that a motorized treadmill was used,
whereas in the present study voluntarily running wheels were
used. Additionally, mesenteric arteries were used in one study
(9) to examine OLETF SOD isoform protein expression rather
than the thoracic aorta, which was used in the present study.
We (48) have also previously shown in the porcine aorta that
treadmill exercise training induces increases in SOD1 expression, and others (24) have shown that exposure of human aortic
endothelial cells to high laminar shear stress, as observed
during exercise, induces increases in SOD1 expression. It is
possible that voluntarily running does not reach the same
intensity stimulus as treadmill exercise training, which signals
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Fig. 8. A: representative Western immunoblots of p-eNOS (Ser1177; top) and

eNOS (bottom) protein expression in endothelial cells scraped from segments
of the thoracic aorta obtained from LSED, OSED, and OPA rats at 13, 20, and
40 wk of age. B: analysis of eNOS and p-eNOS (Ser1177) protein expression in
endothelial cells scraped from segments of the thoracic aorta. Ratios were
calculated for the optical density of eNOS or p-eNOS at 13, 20, or 40 wk over
that of 13-wk-old rats in each respective group and are expressed as the ratio
of p-eNOS to eNOS. Values are means ' SE of 6 8 determinations. *P %
0.05 vs. 13 wk in each respective group; #P % 0.05 vs. 20 wk in each
respective group.
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changes in aortic SOD isoform expression in endothelium

and/or smooth muscle. Future studies should use a more
rigorous and formal exercise training program to fully elucidate whether aortic SOD isoform expression can indeed be
enhanced by more vigorous exercise during T2DM progression
in the OLETF rat. On the other hand, 20-wk-old sedentary
OLETF rats demonstrated a significant temporal reduction in
SOD2 expression compared with 13-wk-old sedentary OLETF
rats that returned to 13-wk-old levels in 40-wk-old sedentary
OLETF rats. Perhaps the initial decrease in ACh-induced EDR
in the 20-wk-old sedentary OLETF aorta was due to this
temporal reduction in SOD2 not seen in sedentary LETO rats
or physically active OLETF rats, as exercise-induced increases
in SOD2 activity have been implicated to contribute to beneficial cardiac (57) and aortic (58) adaptations in rodents.
Alterations in aortic eNOS protein expression do not appear
to account for the temporal maintenance of endothelial function in physically active OLETF and sedentary LETO rat
abdominal aortas (Fig. 7A). Additionally, the temporal loss of
ACh-induced EDR in sedentary OLETF rats does not appear to
be due to altered eNOS protein content (Fig. 7A). Previous
studies (31, 43) in OLETF rats have yielded similar results,
where neither aortic eNOS protein expression nor basal NO
production were different between diabetic OLETF and nondiabetic LETO controls. On the other hand, Ser1177 p-eNOS
levels at 40 wk in both sedentary LETO and physically active

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AJP-Heart Circ Physiol VOL

body fat (Table 1) and serum parameters as well as significantly higher NO production (Table 2), thereby preventing the
development of T2DM and metabolic syndrome in OLETF
rats. Taken together, these data indicate that physical activity in
OLETF rats likely contributed directly or indirectly to their
ability to temporally maintain ACh-induced EDR in their
abdominal aortas via an increase in the aortic p-eNOS/eNOS
ratio and NO production.
Our results suggest that a NOS- and COX-independent
mediator partially compensated for the initial decrease in
ACh-induced EDR in the 20-wk-old sedentary OLETF rat
aorta. This interpretation was inferred from the results in rings
pretreated with the NOS inhibitor L-NNA plus the COX inhibitor Indo (L-NNA ! Indo; Fig. 2F). When used in combination,
L-NNA and Indo blocked the two well-established EDR signaling pathways resulting from NOS and COX activity. Data
obtained during this treatment of the aortas demonstrated that
from 13 to 20 wk sedentary OLETF rat abdominal aortas
exhibited a significant temporal increase in endothelium NOSand COX-independent-mediated relaxation signaled by ACh
(i.e., increased contribution from another mediator of EDR,
perhaps EDHF), which returned to 13-wk levels by 40 wk. The
temporal increase in EDHF-dependent ACh-induced EDR coincided with a 20 35% decrease in ACh-induced EDR from 13
to 20 wk in sedentary OLETF rats (Fig. 1E). Temporal changes
in EDHF-dependent ACh-induced EDR were not observed in
sedentary LETO or physically active OLETF rat groups, and
the maximal EDHF-dependent ACh-induced EDR achieved
was 10 20% less than that of sedentary OLETF rats (Fig. 2, B
and D). Several studies in OLETF rats appear to conflict with
our findings. Matsumoto et al. (33, 34) reported that at 60 wk
of age OLETF rat mesenteric arteries and Minami et al. (25,
43) reported that at 35 40 wk of age in OLETF thoracic aortas
that EDHF-dependent ACh-induced EDR responses are significantly depressed/absent compared with LETO controls. However, these studies did not make comparisons of vascular
function within OLETF rat groups while the animals aged, as
we did in the present study; instead, observations were made at
a single age and not throughout the progression of T2DM.
Further studies are warranted to investigate EDHF-dependent
ACh-induced EDR responses in the OLETF rat using the same
vessels from animals of the same age and that examine AChinduced EDR responses during the progression of T2DM to
better comprehend these conflicting results.
There was a significant temporal decrease in the sensitivity
of the abdominal aorta to SNP from 20 to 40 wk in physically
active OLETF rats and sedentary OLETF rats (Fig. 3, C and
D); however, no such change in sensitivity was seen in 40-wkold sedentary LETO animals (Fig. 3A). This is the first report
where decreased sensitivity to NO was observed in the OLETF
rat vasculature. Treatment with Indo returned this decrease in
sensitivity in 40-wk-old physically active OLETF rats back to
13- and 20-wk-old levels (Fig. 4C) but not in 40-wk-old
sedentary OLETF rats (Fig. 4E). This difference between the
responses of physically active OLETF rats and sedentary
OLETF rats likely indicates that COX-generated metabolites
contribute to the decreased NO sensitivity in 40-wk-old physically active OLETF rat aortas. COX enzymes are known to
metabolize arachidonic acid (8), producing metabolites that
have been shown to enhance mesenteric artery and skeletal
muscle arteriole vascular smooth muscle contractility in rat
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OLETF rat groups were significantly higher than those in 13and 20-wk-old rats in each respective group (Fig. 7B). Further
analysis of the p-eNOS/eNOS ratio shown in Fig. 8, A and B,
revealed that relative to the total pool of available eNOS in the
thoracic aorta endothelium, the amount of p-eNOS was significantly greater in 40-wk-old rats compared with 13- and 20wk-old rats in the sedentary LETO and physically active
OLETF rat groups. This is the first report to demonstrate that
physical activity in an obese T2DM model results in temporal
increases in both aortic p-eNOS protein expression and the
p-eNOS/eNOS ratio with a similar concurrent increase in
plasma NOx concentration back to nondiabetic LETO control
levels (Table 2). The temporal increase in p-eNOS at 40 wk in
sedentary LETO and physically active OLETF rat aortas could
serve as a compensatory mechanism associated with aging in
these rats that is not present in sedentary diabetic OLETF rats.
Matsumoto et al. reported similar reductions in p-eNOS protein
content in the mesenteric arteries of sedentary OLETF rats
compared with LETO control rats (33) and recently showed
that OLETF rats aged similarly to those in the present study
had significantly lower p-eNOS/eNOS ratios than LETO rats
(36). Our findings are also in agreement with recent studies in
humans (17) and rodents (19, 60) that demonstrated that
exercise training resulted in a marked increase in the phosphorylation of eNOS at Ser1177. Collectively, these findings suggest
that in physically active OLETF and LETO rats, p-eNOS
expression increases with age in the aortic endothelium, perhaps due to changes in the hemodynamic signals produced by
aging and physical activity levels in OLETF rats. The reason
that sedentary OLETF rat aortic endothelium p-eNOS expression does not increase similarly with aging may be related to
physical activity levels, obesity, and/or T2DM progression.
These conclusions are also in agreement with the current
literature, which suggests that the mechanical stimulus of
chronically elevated levels of systemic shear stress in response
to regular physical activity in OLETF rats compared with
sedentary OLETF rats would account, in part, for the normalized p-eNOS levels seen in physical active OLETF rats. Reports on the influence of shear stress from in vivo human
laboratories (13, 14, 52) and from laboratories that use cultured
endothelial cells (20, 21, 55, 56) highlight the importance of
episodic increases in shear rate for maintaining normal endothelial function via the eNOS/p-eNOS signaling pathway.
However, additional studies are needed to fully elucidate the
exact aging effect that interacts with physical inactivity in
sedentary OLETF rats.
Hyperphagia and physical inactivity in sedentary OLETF
rats resulted in increased percent body fat (Table 1) and
elevated glucose, HbA1c, and triglyceride levels (Table 2)
compared with sedentary LETO rats and physically active
OLETF rats at each age during T2DM disease progression.
Increased adiposity and elevated triglycerides are well-established risk factors for endothelial dysfunction, independent of
T2DM (53, 54). These factors, which are present in metabolic
syndrome (15, 61), likely contributed to the inability of sedentary OLETF rats to temporally maintain ACh-induced EDR
in their abdominal aortas in addition to the lack of an increase
in the aortic p-eNOS/eNOS ratio and significantly lower NO
production. Conversely, the absence of a genetic predisposition
to T2DM in sedentary LETO rats and regular physical activity
in physically active OLETF rats resulted in a lower percent

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ACKNOWLEDGMENTS
OLETF and LETO rats were a generous gift of Tokushima Research
Institute, Otsuka Pharmaceutical (Tokushima, Japan). The authors thank Whitney Collins, Ann Melloh, Pam Thorne, Miles Tanner, and Grace Uptergrove
for technical assistance. The authors also acknowledge Dr. Richard Madsen for
conducting statistical analysis of the data presented in this article.
GRANTS
This work was partially supported by the College of Veterinary Medicine
and Department of Internal Medicine of the University of Missouri as well as
by National Institutes of Health Grants HL-36088 (to M. H. Laughlin) and
F32-DK-83182 (to R. S. Rector). This work was also supported with resources
and the use of facilities at the Harry S. Truman Memorial Veterans Hospital in
Columbia, MO.
DISCLOSURES
No conflicts of interest are declared by the author(s).
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to contribute to depressed ACh-induced relaxation in OLETF
mesenteric arteries (36) via increases in the production of vasoconstrictor COX-derived metabolites. Collectively, these studies
and our data suggest that overproduction of COX metabolites may
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responsiveness in a sedentary lifestyle and T2DM progression,
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In conclusion, we have shown that a sedentary lifestyle in
the OLETF rat results in a temporal decrease of abdominal
aorta ACh-induced EDR and that ACh-induced EDR is temporally maintained in OLETF rats that engage in regular
physical activity. Our data also indicate that temporal alterations in aortic SOD isoforms are not the putative underlying
mechanisms for the temporal decline in sedentary OLETF rat
ACh-induced aortic EDR. However, the lack of an increase in
the aortic p-eNOS/eNOS ratio and plasma NOx concentration
in 40-wk-old sedentary OLETF rats that was observed in
physically active OLETF rats and sedentary LETO rats at 40
wk likely contributed to the observed decline in sedentary
OLETF rat abdominal aortic ACh-induced EDR. This is the
first report showing that regular physical activity on voluntary
running wheels in conjunction with aging in the OLETF rat
results in a temporal increase in aortic p-eNOS expression
compared with sedentary OLETF rats. Therefore, we propose
an important role for p-eNOS in temporally maintaining
OLETF aortic endothelial function that can be exploited by the
prevention of T2DM and obesity through regular physical
activity.

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