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OR I G I N A L A R T I C L E doi: 10.1111/j.1463-1326.2007.00814.x
Vascular and neural dysfunction in Zucker diabetic fatty rats:
a difficult condition to reverse
C. L. Oltman,1,2 E. P. Davidson,1,2 L. J. Coppey,1,2 T. L. Kleinschmidt,1 D. D. Lund,1,2
E. T. Adebara2 and M. A. Yorek1,2
1Veteran Affairs Medical Center, University of Iowa, Iowa City, IA, USA2Department of Internal Medicine, University of Iowa, Iowa City, IA, USA
Aim: We had previously demonstrated that vascular and neural dysfunction in Zucker diabetic fatty (ZDF) rats is
progressive. In this study, we sought to determine whether monotherapy of ZDF rats can reverse the vascular and nerve
defects.
Methods: ZDF rats at 16 weeks of age were treated for 12 weeks with the angiotensin-converting enzyme inhibitor
enalapril, the antioxidant a-lipoic acid, the HMG-CoA reductase inhibitor rosuvastatin or the PPARg agonist rosigli-
tazone. Vasodilation of epineurial arterioles was measured by videomicroscopy. Endoneurial blood flow (EBF) was
measured by hydrogen clearance, and nerve conduction velocity was measured following electrical stimulation of
motor or sensory nerves.
Results: Motor nerve conduction velocity (MNCV), sensory nerve conduction velocity (SNCV) (70 and 77% of control,
respectively), EBF (64% of control), and vascular relaxation in response to acetylcholine (50% of control) and calci-
tonin gene-related peptide (CGRP; 73% of control) are impaired in ZDF rats at 28 weeks of age compared with lean
littermate controls. Treatment with enalapril and a-lipoic acid attenuated the decrease in MNCV and SNCV. Enalapril,
a-lipoic acid and rosiglitazone treatment of ZDF rats were partially effective in improving endothelium-dependent
vascular dysfunction as measured by vascular relaxation in response to acetylcholine. The same drugs also attenuated
the decrease in EBF. However, impairment in vascular relaxation in response to CGRPwas improvedwith only a-lipoicacid or rosuvastatin treatment. The increase in superoxide and nitrotyrosine levels in vascular tissue was attenuated by
all treatments.
Conclusions: The efficacy of monotherapy treatment of ZDF rats using different classes of drugs for vascular and
neural dysfunction once complications have developed did not achieve expected levels. This could be because of the
complex aetiology of vascular and neural disease in type 2 diabetes.
Keywords: metabolic syndrome, neuropathy, obesity, oxidative stress, reactive oxygen species, vascular reactivity
Received 21 January 2007; accepted 26 February 2007
Introduction
Diabetic neuropathy is a debilitating disorder that occurs
in about 50% of patients with diabetes [1]. Neuropathy is
a late finding in type 1 diabetes but can be an early find-
ing in type 2 diabetes and frequently is the presenting
symptom in type 2 diabetes [1,2]. Evidence suggests
that there are at least five major pathways involved in
the development of diabetic neuropathy: metabolic,
vascular, immunological, neurohormonal growth factor
Correspondence:
Mark A. Yorek, Building 40, Room 204,Veteran Affairs Medical Center, Iowa City, IA 52246, USA.
E-mail:
64 j Diabetes, Obesity and Metabolism, 10, 2008, 64–74# 2007 The Authors
Journal Compilation # 2007 Blackwell Publishing Ltd
deficiency and extracellular matrix remodelling [2].
There is also evidence that the aetiology and pathology
of diabetic neuropathy in type 1 and type 2 diabetes
may be different [3–5]. In light of the complicated aetiol-
ogies, an effective treatment for diabetic neuropathy has
not yet been identified.
What is surprising is the small number of preclinical
studies that have been performed examining potential
treatments for diabetic neuropathy in animal models of
type 2 diabetes. Whereas there have been a large number
of studies, primarily using streptozotocin-induced dia-
betic rats, examining treatment paradigms for diabetic
neuropathy for type 1 diabetes, there are relatively few
of these studies with type 2 diabetes models.
Zucker diabetic fatty (ZDF) rats are an animalmodel for
type 2 diabetes. In this model, all fatty males become
hyperglycaemic by 8 weeks of age and glucose remains
elevated through their lifespan [6]. Initially, ZDF rats are
hyperinsulinaemic. However, by 22–42 weeks of age,
serum insulin levels decline to below the levels of insu-
lin in age-matched lean control rats [6]. A similar char-
acteristic is seen in human type 2 diabetes, which is
thought to be caused by pancreas/b-cell exhaustion.
Free fatty acids, triglycerides and cholesterol levels are
significantly higher in ZDF diabetic rats throughout
their lifespan compared with lean littermate controls
[6]. Previously, we have demonstrated that development
of microvascular and nerve dysfunction in ZDF rats is
progressive with impairment of acetylcholine-mediated
vascular relaxation occurring prior to nerve blood flow
and conduction deficits [7]. At 6 weeks of age, these
ZDF rats were hyperinsulinaemic but had a normal
blood glucose level. However, by 8–10 weeks of age, the
rats became hyperglycaemic and relaxation by epi-
neurial arterioles to acetylcholine was impaired [7].
Motor nerve conduction velocity (MNCV) decreased at
12–14 weeks of age and endoneurial blood flow (EBF)
impaired at 24 weeks of age [7]. In this study, we sought
to determine whether microvascular dysfunction and
slowing of nerve conduction velocity could be
improved by treatment with optimal doses of enalapril,
a-lipoic acid, rosuvastatin or rosiglitazone.
Methods
Unless statedotherwise, all chemicalsused in these studies
were obtained from Sigma Chemical (St Louis, MO, USA).
Animals
Male ZDF and lean (control) rats 6 weeks of age were
obtained from Charles River Laboratories (Wilmington,
MA,USA). The animalswere housed in a certified animal
care facility and food [7001 (for lean rats) and 7013 (for
ZDF rats); Harlan Teklad, Madison, WI, USA] and water
were provided ad libitum. All institutional (approval
Animal Care and Use Review Form (ACURF) 0290608)
and National Institutes of Health guidelines for use of
animals were followed. After 16 weeks of age, the rats
were used for studies described below. The ZDF rats
were divided into five groups. One group was fed the
unsupplemented 7013 diet; the second group was fed
the 7013 diet containing 400 mg/kg enalapril; the third
group was fed the 7013 diet containing 2.5 g/kg a-lipoicacid; the fourth group was fed the 7013 diet containing
500 mg/kg rosuvastatin and the fifth group was fed the
7013 diet containing 50 mg/kg rosiglitazone. The aver-
age amount of chow consumed by ZDF rats was 50 g/
day/kg rat. Thus, the respective groups of rats received
approximately 20 mg/kg rat of enalapril, 125 mg/kg rat
of a-lipoic acid, 25 mg/kg rat of rosuvastatin and
2.5 mg/kg rat of rosiglitazone per day. The supple-
mented diets were prepared from the meal form of the
diet. The drugs were thoroughly mixed into the meal by
stirring for 1 h. Afterwards, the diets were pelleted and
dried in a vacuum oven set at 40 °C overnight. The
unsupplemented diets for the lean (7001) and untreated
ZDF rats (7013) were also prepared from the meal. The
treatment period lasted for 12 weeks.
Thermal Nociceptive Response
The day before the terminal studies, thermal nociceptive
response in the hindpaw was measured using the Har-
greaves method with instrumentation provided by IITC
Life Science (Woodland Hills, CA, USA; model 390G).
The rat was placed in the observation chamber on top of
the thermal testing apparatus and allowed to acclimate to
the warmed glass surface (30 °C) and surroundings for
a period of 15 min. The mobile heat source was man-
oeuvred so that it was under the heel of the hindpaw
and then activated, a process that activates a timer and
locallywarms the glass surface.When the ratwithdrew its
paw, the timer and the heat source were turned off [8].
Following an initial recording, which was discarded,
four measurements were made for each hindpaw, with
a rest period of 5 min between each set of measure-
ments. The mean of the measurements, reported in sec-
onds, were used as a measure of the thermal nociceptive
response.
On the day of the experiment, rats were anaesthetized
with Nembutal i.p. (50 mg/kg, i.p.; Abbott Laboratories,
North Chicago, IL, USA) and non-fasting blood glucose
levels were determined with the use of glucose oxidase
C. L. Oltman et al. Vascular and neural dysfunction in ZDF rats j OA
# 2007 The Authors
Journal Compilation # 2007 Blackwell Publishing LtdDiabetes, Obesity and Metabolism, 10, 2008, 64–74 j 65
reagent strips (LifeScan, Milpitas, CA, USA). Serum
samples were collected for determination of serum-free
fatty acid, triglyceride and free cholesterol using commer-
cial kits from Roche Diagnostics (Mannheim, Germany),
Sigma Chemical and BioVision (Mountain View, CA,
USA) respectively. For these analyses, themanufacturer’s
instructions were followed. Afterwards, MNCV, SNCV
and EBF in the sciatic nerve were determined and tissue
containing the epineurial arterioles was collected.
MNCV and SNCV
MNCV was determined as previously described using
a non-invasive procedure in the sciatic-posterior tibial
conducting system [9–12]. SNCV was determined using
the digital nerve to the second toe as described by Obro-
sova et al. [13]. The MNCV and SNCV were reported in
metres per second.
Endoneurial Blood Flow
Sciatic nerve EBF was determined as previously
described using the hydrogen clearance method [9–12].
The hydrogen clearance data were fitted to a mono- or
bi-exponential curve using commercial software (PRISM;
GraphPad, San Diego, CA, USA). Nutritive blood flow
(ml/min/100 g) was calculated using the equation
described by Young [14], and vascular conductance (ml/
min/100 g/mmHg) was determined by dividing nutritive
blood flow by the average mean arterial blood pressure.
Vascular Reactivity
Videomicroscopy was used to investigate in vitro vaso-
dilatory responsiveness of arterioles vascularizing the
region of the sciatic nerve as previously described [9–
12]. Cumulative concentration–response relationships
were evaluated for acetylcholine (10�8–10�4 M) and cal-
citonin gene-related peptide (CGRP) (10�11–10�8 M)
using vessels from each group of rats. At the end of
each dose–response determination, a maximal dose of
sodium nitroprusside (10�4 M) was added. Afterwards,
papaverine (10�5 M) was added to determine maximal
vasodilation, which was consistently the same as the
vascular tone of the resting vessel at 40 mmHg.
Detection of Superoxide
Hydroethidine (Molecular Probes, Eugene, OR, USA), an
oxidative fluorescent dye, was used to evaluate in situ
levels of superoxide ðO �2 Þ in epineurial vessels as
described previously [9,11]. This method provides sen-
sitive detection of ðO �2 Þ. Vessel segments from 28-week-
old lean rats and ZDF untreated and treated rats were
processed and imaged in parallel. The labelled vessels
derived from these studies were visualized with a Zeiss
LSM 510 laser scanning confocal microscope using
40� objectives. Laser settings were identical for acqui-
sition of all images. Superoxide levels using the aorta
were also measured by lucigenin-enhanced chem-
iluminescence as described previously [15]. Relative
light units (RLU) were measured using a Zylux FB12
luminometer. Background activity was determined and
subtracted, and RLU was normalized to surface area.
Oneof the twomechanismsbywhich acetylcholine can
mediate vascular relaxation in arterioles that provide cir-
culation to the sciatic nerve is through the production of
nitric oxide [16]. The chemistry of nitric oxide is com-
plex, and several biochemical pathways other than
nitric oxide production can influence nitric oxide bio-
activity. For example, superoxide anion can interact
with nitric oxide to form peroxynitrite [17]. This reac-
tion reduces the efficacy of nitric oxide to act as a signal
transduction agent. Peroxynitrite is a highly reactive
intermediate known to nitrate protein tyrosine residues
and causes cellular oxidative damage [17,18]. To deter-
mine whether formation of superoxide by arterioles that
provide circulation to the sciatic nerve promotes the for-
mation of peroxynitrite, we measured 3-nitrotyrosine
(3-nitrotyrosine is a stable biomarker of tissue peroxyni-
trite formation) in vessel sections. Briefly, frozen tissue
segments of arterioles were cut into 5 mm sections and
then incubated in phosphate-buffered saline solution
containing 1% Triton X-100 and 0.1% bovine serum
albumin for 30 min at room temperature. Afterwards,
the samples were incubated in this buffer solution con-
taining mouse antinitrotyrosine antibody (Upstate, Lake
Placid, NY, USA) overnight at 4 °C. After washing, the
sections were incubated at room temperature for 2 h
with Alexa Fluor 555 goat antimouse IgG (Molecular
Probes). Sections were then rinsed and mounted with
VectorShield. The labelled vessels derived from these
studies were visualized with an Olympus IX71 inverted
research microscope interfaced with a personal com-
puter (PC) containing SIMPLEPCI imaging software.
Pixel intensity for superoxide or nitrotyrosine immu-
nostaining was determined for each vessel segment and
averaged for each condition. Data were reported for RLU
per mm2.
Data Analysis
The results are presented as mean � s.e.m. Comparisons
between the groups for body weight, blood pressure,
OA j Vascular and neural dysfunction in ZDF rats C. L. Oltman et al.
66 j Diabetes, Obesity and Metabolism, 10, 2008, 64–74# 2007 The Authors
Journal Compilation # 2007 Blackwell Publishing Ltd
blood glucose, MNCV, SNCV, EBF, thermal nociception,
serum-free fatty acid, triglyceride, cholesterol, and aorta
and epineurial arteriole superoxide levels were con-
ducted using a one-way ANOVA and Newman–Keuls test
for multiple comparisons and the Bonferroni–Dunn test
(PRISM software; GraphPad). Concentration–response
curves for acetylcholine- and CGRP-induced relaxation
were compared using a two-way repeated measures
ANOVA with autoregressive covariance structure using
the PROC MIXED program of SAS (SAS Institute, Cary,
NC, USA) [9–12]. Whenever significant interactions
were noted, specific treatment–dose effects were ana-
lysed using a Bonferroni–Dunn test. A p value of less
than 0.05 was considered significant.
Results
Changes in Body Weight, Blood Pressure, Blood
Glucose and Serum Lipid Levels
Table 1 presents data for important endpoints of the
study. At 16 weeks of age, the weight and blood glucose
level for lean and ZDF rats were 376 � 5 g and
53 � 1 mg/dl, and 395 � 5 g and 248 � 6 mg/dl respec-
tively. At 28 weeks of age, untreated ZDF rats weighed
less than lean rats. ZDF rats treated with a-lipoic acid or
rosuvastatin also weighed less than the lean rats but were
not different than the ZDF untreated rats. The weight of
ZDF rats treated with enalapril or rosiglitazone was not
different from lean rats. However, ZDF rats treated with
rosiglitazone weighed significantly more than untreated
ZDF rats. Blood glucose levels were significantly
increased to 350% in ZDF rats compared with lean rats.
Treatment of ZDF rats did not change blood glucose lev-
els; however, there was a trend for treatment with a-lipoic acid (p < 0.10) to lower blood glucose level. The
per cent difference in blood glucose levels between
untreated ZDF rats and ZDF rats treated with enalapril,
lipoic acid, rosuvastatin and rosiglitazone was 105, 83,
94 and 91% respectively. Blood pressure was not signifi-
cantly increased in ZDF rats compared with lean rats (p <
0.30, 109% of control). However, blood pressure was
significantly decreased in enalapril-treated ZDF rats
compared with untreated ZDF rats (82%). The other treat-
ments did not significantly change blood pressure record-
ings between treated and untreated ZDF rats [lipoic acid
(98% of untreated ZDF rats), rosuvastatin (88% of un-
treated ZDF rats) and rosiglitazone (94% of untreated
ZDF rats)]. Serum cholesterol, triglyceride and free fatty
acid levels were significantly increased in untreated
ZDF rats (269, 313 and 252% respectively). Treatment
of ZDF rats with a-lipoic acid (69% of untreated ZDF rats,
p < 0.40) or rosuvastatin (56% of untreated ZDF rats,
p < 0.20) lowered serum triglyceride levels but neither of
these nor the other treatments changed serum cholesterol
or free fatty acid levels.
Changes in Nerve Conduction Velocity and Thermal
Nociception
At 28 weeks of age, MNCV and SNCV were significantly
decreased in untreated ZDF rats compared with lean rats
(70 and 77% of control respectively) (figure 1). Treating
ZDF rats with enalapril attenuated the decrease in
MNCV and SNCV (85 and 94% of control respectively).
Treatment of ZDF rats with a-lipoic acid also reduced
the slowing of nerve conduction velocity (82 and 89%
of control respectively). However, a-lipoic acid was less
Table 1 Effect of treatment of ZDF rats with enalapril, a-lipoic acid, rosuvastatin or rosiglitazone on body weight, blood
pressure, blood glucose, serumcholesterol, triglycerides, free fatty acids, superoxide levels in the aorta and thermal nociception
Determination Lean (12)
ZDF
untreated (9)
ZDF
Enalapril (8)
ZDF lipoic
acid (8)
ZDF
rosuvastatin (9)
ZDF
rosiglitazone (8)
Final weight (g) 453 � 11 397 � 10* 428 � 14 368 � 11* 396 � 20* 481 � 24yBlood pressure (mmHg) 120 � 7 131 � 5 108 � 5* 129 � 5 115 � 6 123 � 4
Blood glucose (mg/dl) 85 � 5 311 � 22* 326 � 28* 257 � 16* 293 � 12* 283 � 20*
Cholesterol (mg/ml) 0.61 � 0.03 1.64 � 0.28* 1.50 � 0.30* 1.78 � 0.16* 2.22 � 0.26* 2.20 � 0.32*
Triglycerides (mg/ml) 8.9 � 0.7 27.9 � 8.1* 25.9 � 5.6* 19.3 � 5.4 15.6 � 2.6 31.0 � 7.0*
Free fatty acids (mmol/l) 0.23 � 0.03 0.58 � 0.08* 0.64 � 0.05* 0.67 � 0.07* 0.63 � 0.08* 0.50 � 0.06*
Aorta superoxide (RLU) 2.11 � 0.20 3.45 � 0.28* 2.09 � 0.23y 2.59 � 0.31y 2.17 � 0.38y 1.93 � 0.17yThermal nociception (s) 9.5 � 0.2 17.4 � 1.3* 10.7 � 0.6y 10.9 � 0.3y 12.3 � 0.6y 14.3 � 1.6*
Numbers in parentheses indicate the number of experimental animals.
Data are presented as the mean � s.e.m.
RLU, relative light units; ZDF, Zucker diabetic fatty.
*p < 0.05 compared with lean rats.
yp < 0.05 compared with ZDF untreated rats.
C. L. Oltman et al. Vascular and neural dysfunction in ZDF rats j OA
# 2007 The Authors
Journal Compilation # 2007 Blackwell Publishing LtdDiabetes, Obesity and Metabolism, 10, 2008, 64–74 j 67
efficacious than enalapril as MNCV and SNCV
remained significantly decreased compared with lean
rats. In contrast, treatment of ZDF rats with rosuvastatin
(77 and 83% of control, respectively) or rosiglitazone
(79 and 80% of control, respectively) did not prevent
the decrease in MNCV and SNCV.
ZDF rats at 28 weeks of agewere hypoalgesic compared
with lean rats (183%of control) (table 1). ZDF rats treated
with enalapril, a-lipoic acid or rosuvastatin had greater
thermal nociception than untreated ZDF rats (113, 115
and 129% of control respectively). ZDF rats treated with
rosiglitazone had higher values of thermal nociception
(151% of control).
Changes in Nerve Blood Flow and Vascular
Relaxation
EBF was significantly decreased in ZDF rats at 28 weeks
of age comparedwith lean rats (64%of control) (figure 2).
Treatment of ZDF rats with a-lipoic acid attenuated the
decrease in EBF (143% of control). Treatment of ZDF
rats with enalapril or rosiglitazone was also efficacious
in attenuating the decrease in EBF but because of
increased variability, the differences were not signifi-
cant compared with untreated ZDF rats [p < 0.30 (105%
of control) and p < 0.20 (101% of control) respectively].
Treating ZDF rats with rosuvastatin did not prevent the
decrease in EBF (43% of control).
We have previously reported that vascular relaxation
in response to acetylcholine or CGRP was impaired at 8
and 28 weeks of age, respectively, in ZDF rats [7]. Data
in figure 3 demonstrate that treating ZDF rats with
enalapril and to a lesser extent with a-lipoic acid or
rosiglitazone significantly improved vascular dys-
function in epineurial arterioles. Treating ZDF rats
with rosuvastatin did not improve acetylcholine-
mediated vascular relaxation. Epineurial arterioles of
the sciatic nerve are innervated by sensory nerves that
contain CGRP [19]. Moreover, CGRP is a potent vaso-
dilator in epineurial arterioles, and CGRP-mediated
vasodilation is impaired in epineurial arterioles from
either type 1 or type 2 diabetic rats [7,19]. Data in
figure 4 demonstrate that treating ZDF rats with
a-lipoic acid or rosuvastatin partially attenuated the
impairment in CGRP-mediated relaxation. In contrast,
treating ZDF rats with enalapril or rosiglitazone did
not prevent impairment in CGRP-mediated vascular
relaxation.
Changes in Markers of Oxidative Stress
Increase in oxidative stress has been widely implicated
in the development of vascular dysfunction in diabetes
[20]. Data in table 1 demonstrate that superoxide levels
are increased in the aortas of untreated ZDF rats at
28 weeks of age compared with lean rats (164% of
Lean (12) ZDF ZDF ZDF ZDF ZDF
MN
CV
(m/s
)
10
20
30
40
50
60
70
SNC
V (m
/s)
10
20
30
40
50
60
70
untreated (9) enalapril (8) lipoic Acid (8) rosuvastatin (9) rosiglitazone (8)
*
*
††
*
†
*
†**
* *
Fig. 1 Determination of the effect of a 12-week treatment with enalapril, a-lipoic acid, rosuvastatin or rosiglitazone on
motor nerve conduction velocity (MNCV) and sensory nerve conduction velocity (SNCV) in 16-week-old Zucker diabetic
fatty (ZDF) rats. Data are presented as the mean � s.e.m. in m/s. The number of experimental determinations is presented
in parentheses. *p < 0.05 compared with lean rats; yp < 0.05 compared with untreated ZDF rats.
OA j Vascular and neural dysfunction in ZDF rats C. L. Oltman et al.
68 j Diabetes, Obesity and Metabolism, 10, 2008, 64–74# 2007 The Authors
Journal Compilation # 2007 Blackwell Publishing Ltd
control). Treating ZDF rats with enalapril, a-lipoic acid,
rosuvastatin or rosiglitazone significantly reduced
superoxide levels in the aorta (99, 123, 103 and 91% of
control respectively). Data in figure 5 demonstrate that
superoxide and nitrotyrosine levels are increased in epi-
neurial arterioles from untreated ZDF rats compared
with lean rats (178 and 187% of control respectively).
Compared with untreated ZDF rats, treatment with
Lean (12) ZDF ZDF ZDF ZDF ZDF
EBF
nutri
tive
(ml/m
in/1
00 g
)
5
10
15
20
25
30
35
EBF
cond
ucta
nce
(ml/m
in/1
00 g
/mm
Hg)
0.05
0.10
0.15
0.20
0.25
0.30
untreated (9) enalapril (8) lipoic Acid (8) rosuvastatin (9) rosiglitazone (8)
*
†
*
*
†
*
Fig. 2 Determination of the effect of treatment of Zucker diabetic fatty (ZDF) rats with enalapril, a-lipoic acid, rosuvastatin
or rosiglitazone for 12 weeks at 16 weeks of age on endoneurial blood flow (EBF). Data are presented as the mean � s.e.m.
for nutritive flow (ml/min/100 g) and conductance (ml/min/100 g/mmHg). The number of experimental determinations is
presented in parentheses. *p < 0.05 compared with lean rats; yp < 0.05 compared with untreated ZDF rats.
Acetylcholine (M)
1e–8 1e–7 1e–6 1e–5 1e–4
% R
ela
xation
0
20
40
60
80
100Lean (16)ZDF untreated (11)ZDF enalapril (11)ZDF lipoic Acid (12)ZDF rosuvastatin (12)ZDF rosiglitazone (15)
**
*
*
*
*†
*
**
†*
†
*
†
***
†*††
Fig. 3 Determination of the effect of treatment of Zucker diabetic fatty (ZDF) rats with enalapril, a-lipoic acid, rosuvastatin
or rosiglitazone for 12 weeks at 16 weeks of age on acetylcholine-mediated vascular relaxation of epineurial arterioles of
the sciatic nerve. Pressurized arterioles (40 mmHg) were constricted with U46619 (30–50%) and incremental doses of ace-
tylcholine were added to the bathing solution while recording steady-state vessel diameter. Data are presented as the mean
of % relaxation � s.e.m. The number of experimental determinations is presented in parentheses. *p < 0.05 compared
with lean rats; yp < 0.05 compared with untreated ZDF rats.
C. L. Oltman et al. Vascular and neural dysfunction in ZDF rats j OA
# 2007 The Authors
Journal Compilation # 2007 Blackwell Publishing LtdDiabetes, Obesity and Metabolism, 10, 2008, 64–74 j 69
enalapril (101 and 119% of control, respectively), a-lipoic acid (147 and 72% of control, respectively) or
rosuvastatin (97 and 125% of control, respectively) sig-
nificantly reduced superoxide and nitrotyrosine levels
in epineurial arterioles.
Discussion
Comparedwith studies using type 1diabetic animalmod-
els, few preclinical pharmacological intervention studies
for microvascular and neural complications have been
performed for type 2 diabetes. As the aetiology and
pathology of neuropathy in type 2 diabetes may be differ-
ent, there is clearly a need for preclinical studies to assess
the outcome of pharmacological treatment [3–5]. This
study evaluated the effects of several interventions on
measure of diabetic vascular and neural complications
in ZDF rats.
Based on our previous studies with streptozotocin-
induced diabetic rats and a review of the literature, we
decided to study the effects of enalapril (angiotensin-
converting enzyme inhibitor), a-lipoic acid (antioxidant),
rosuvastatin (HMG-CoA reductase inhibitor) and rosigli-
tazone (PPARg agonist) [9,12,21–23]. Each of these drugs
has been used by us or others in animal studies for treat-
ment of vascular and neural dysfunction in type 1 dia-
betes, nephropathy in type 2 diabetes or hypertension
[9,12,21–24]. All drugs were delivered in the diet, and
the doses were based on our previous studies or the lit-
erature [9,12,21–23].
Our previous studies with ZDF rats demonstrated that
vascular and neural complications are progressive [7].
Vascular dysfunction appeared within 8 weeks of age
followed by decreased nerve conduction velocity (12–
14 weeks of age) and blood flow (24 weeks of age) [7].
As many patients are not diagnosed with type 2 diabetes
until they develop symptoms of complications, we
decided to initiate treatment in the rats at 16 weeks of
age, similar to the time course observed in humans [2].
At 16 weeks of age, ZDF rats were hyperglycaemic for
8 weeks and vascular and neural complications have
developed [7]. Studies have been carried out with ZDF
rats demonstrating that treatments with several different
drugs can prevent the onset of hyperglycaemia. However,
this is not how type 2 diabetic patients are clinically trea-
ted for complications, and no preclinical studies are
available addressing the reversal or delaying progression
of complications once intervention therapy has been star-
ted. The treatment period was 12 weeks. Based on stud-
ies we performed with streptozotocin-induced diabetic
CGRP (M)
1e–11 1e–10 1e–9 1e–8
% R
elax
atio
n
0
20
40
60
80
100Lean (15)ZDF untreated (14)ZDF enalapril (14)ZDF lipoic Acid (12)ZDF rosuvastatin (15)ZDF rosiglitazone (15)
**
***
*
Fig. 4 Determination of the effect of treatment of Zucker diabetic fatty (ZDF) rats with enalapril, a-lipoic acid, rosuvastatin
or rosiglitazone for 12 weeks at 16 weeks of age on CGRP-mediated vascular relaxation of epineurial arterioles of the sciatic
nerve. Pressurized arterioles (40 mmHg) were constricted with U46619 (30–50%) and incremental doses of calcitonin gene-
related peptide (CGRP) were added to the bathing solution while recording steady-state vessel diameter. Data are presented
as the mean of % relaxation � s.e.m. The number of experimental determinations is presented in parentheses. *p < 0.05
compared with lean rats.
OA j Vascular and neural dysfunction in ZDF rats C. L. Oltman et al.
70 j Diabetes, Obesity and Metabolism, 10, 2008, 64–74# 2007 The Authors
Journal Compilation # 2007 Blackwell Publishing Ltd
rats, we decided that this period should be long enough
to demonstrate efficacy [9,10,12].
The key finding of this studywas that treatment of ZDF
rats with enalapril or a-lipoic acid was moderately suc-
cessful in decreasing nerve dysfunction in ZDF rats.
These two drugs along with rosiglitazone attenuated the
impairment of vascular relaxation to acetylcholine and
nerve blood flow. All four treatments improved thermal
nociception and reduced superoxide levels.However, the
overall success of these treatments was only modest, sug-
gesting that more aggressive combination therapies may
be required to successfully treat vascular and neural com-
plications in type 2 diabetes.
Using streptozotocin-induced diabetic rats, we have
previously demonstrated that antioxidant therapy
(a-lipoic acid and M40403) is an effective treatment for
vascular and neural dysfunction in type 1 diabetes [9,10].
In the present study, enalapril and a-lipoic acid were
less effective in reducing vascular dysfunction and
slowing of nerve conduction velocity compared with
streptozotocin-induced diabetic rats [12]. All treatments
were associated with lower superoxide levels in epi-
neurial arterioles or aortas, but this outcome did not
translate to a similar improvement in vascular and neu-
ral function. This suggests that in addition to increased
oxidative stress, other pathological conditions more
prevalent in type 2 than in type 1 diabetes may be con-
tributing to vascular and neural complications.
It was interesting to observe that the three treatment
conditions that improvedacetylcholine-mediatedvascular
Fig. 5 Detection of superoxide and nitrotyrosine in epineurial arterioles of the sciatic nerve from lean (control), untreated
Zucker diabetic fatty (ZDF) and ZDF rats treated with enalapril, a-lipoic acid, or rosuvastatin. Presented are representative
fluorescent photomicrographs of confocal microscopic sections of epineurial arterioles of the sciatic nerve for superoxide
(top) and nitrotyrosine (bottom). Analysis of these images using SIMPLEPCI imaging software is presented for superoxide
(left) and nitrotyrosine (right). The data are presented as relative light units (RLU) and normalized to surface area. The lean
control was arbitrarily assigned a value of 1. *p < 0.05 compared with untreated ZDF rats. These values were obtained
from two different rats and five vessel segments were analysed for each individual rat.
C. L. Oltman et al. Vascular and neural dysfunction in ZDF rats j OA
# 2007 The Authors
Journal Compilation # 2007 Blackwell Publishing LtdDiabetes, Obesity and Metabolism, 10, 2008, 64–74 j 71
relaxation in epineurial arterioles fromZDF rats, enalap-
ril, a-lipoic acid and rosiglitazone, also improved nerve
blood flow. This suggests that endothelium-dependent
vascular relaxation may be linked to the regulation of
nerve blood flow. In contrast, treatment with rosuvasta-
tin and a-lipoic acid improved vascular relaxation of
epineurial arterioles in response to CGRP. However,
rosuvastatin treatment did not affect the decrease in
EBF or nerve conduction velocity. This suggests that
improving CGRP-mediated vascular function is not suf-
ficient by itself for attenuating neural complications in
type 2 diabetes. Treatment of ZDF ratswith rosiglitazone
did not prevent slowing of nerve conduction velocity,
although it did attenuate the decrease in nerve blood
flow suggesting that regulation of nerve blood flow is
not the only factor mediating changes in nerve conduc-
tion velocity.
In ZDF rats at 16 weeks of age, vascular relaxation of
epineurial arterioles in response to a maximum dose of
acetylcholine is about 60% of control and this decreased
to 40% of control by 32 weeks of age [7]. Treatment of
ZDF rats with enalapril appeared to halt the progression
of vascular dysfunction but reversal was not apparent.
Treatment of ZDF rats with a-lipoic acid or rosiglitazone
was less effective than enalapril. These drugs slowed
the progression of vascular dysfunction. As slowing of
MNCV occurs by 12–14 weeks of age in ZDF rats, treat-
ment with enalapril or a-lipoic acid appeared to reverse
this deficit. Furthermore, treatment of ZDF rats with
enalapril, a-lipoic acid or rosiglitazone appeared to
improve the decrease in EBF. All four treatments
improved thermal nociception with enalapril and
a-lipoic acid being more effective than rosuvastatin,
which was more effective than rosiglitazone.
Previously, we demonstrated that enalapril treatment
of streptozotocin-induced diabetes after different dura-
tions of diabetes (0–16 weeks) improved diabetes micro-
vascular and neural dysfunction [12]. In a more recent
study, we have shown that enalapril treatment at
20 weeks of age corrected vascular dysfunction in epi-
neurial arterioles and slowing of nerve conduction
velocity in obese Zucker rats [25]. In obese Zucker rats,
enalapril has also been shown to reverse cardiovascular
complications and attenuate the development of glo-
merular damage [26,27]. In Goto–Kakizaki (GK) rats,
a type 2 diabetic model, enalapril treatment for
12 weeks improved endothelium-dependent relaxation
to acetylcholine by nearly 70% [28]. In our studies, ena-
lapril treatment was not as effective; however, in the
studies with GK rats, treatment was started at 8 weeks of
age [28]. Therefore, when treatment is initiated seems to
be an important factor in the success of that treatment.
Our studies have shown that treatment of streptozoto-
cin-induced diabetic rats with a-lipoic acid prevented
vascular and neural impairment [9]. Here, we demon-
strate that a-lipoic acid treatment of ZDF rats was effica-
cious although to a lesser degree compared with type 1
diabetic rats [9]. In type 2 diabetes and obese Zucker
rats, a-lipoic acid has been shown to improve insulin
resistance [29–31]. In our studies, treatment with a-lipoic acid lowered blood glucose levels and improved
oxidative stress. However, this did not translate to
an impressive improvement in vascular and neural
function.
It has been stated that because of the excellent safety
profile of statins, they should be the treatment of choice
for vascular complications in diabetic patients with
hypercholesterolaemia [32]. Statins have been shown to
reduce glucose- and diabetes-induced oxidative stress
and improve vascular function in animal models of
insulin resistance [33,34]. But, in ZDF rats, treatment
with rosuvastatin did not improve microvascular and
neural dysfunction once these complications had devel-
oped. In contrast to our findings, Masaaki and co-
workers reported that rosuvastatin treatment of db/db
mice restored microcirculation of the sciatic nerve and
reversed diabetic neuropathy [35]. In these studies,
drug treatment was started at 8 weeks of age. In db/db
mice, diabetes complications do not appear until after
8 weeks of age [35]. This also indicates that the duration
of diabetes and when treatment is started may be a limit-
ing factor in the success of the treatment.
PPARg agonists, such as rosiglitazone, have been
widely used in the treatment of type 2 diabetes. In studies
with ZDF rats, treatment with a PPARg agonist from the
prediabetic stage prevented the development of diabetic
complications [36,37]. However, as shown by our stud-
ies, the efficacy of rosiglitazone in improving the slow-
ing of nerve conduction velocity was greatly diminished
once complications had developed.
In summary, these studies have demonstrated that it is
difficult to improve microvascular and neural complica-
tions of diabetes once these complications have devel-
oped. This suggests that more aggressive therapy
utilizing a combination of drugs as well as early interven-
tion may be required to successfully treat diabetic neu-
ropathy in type 2 diabetes.
Acknowledgements
This work was supported by a grant from the Order of the
Amaranth through the American Diabetes Association,
Merit Review Grants from the Veterans Affairs Adminis-
tration to Drs M. A. Y. and C. L. O. and by a National
OA j Vascular and neural dysfunction in ZDF rats C. L. Oltman et al.
72 j Diabetes, Obesity and Metabolism, 10, 2008, 64–74# 2007 The Authors
Journal Compilation # 2007 Blackwell Publishing Ltd
Institute of Diabetes and Digestive and Kidney Diseases
Grant DK073990 from the NIH. The contents of this man-
uscript are solely the responsibility of the authors and do
not necessarily represent the official views of the NIH.
We would also like to extend our appreciation to Merck,
GlaxoSmithKline and AstraZeneca for supplying drugs
for these studies.
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74 j Diabetes, Obesity and Metabolism, 10, 2008, 64–74# 2007 The Authors
Journal Compilation # 2007 Blackwell Publishing Ltd