Vascular and neural dysfunction in Zucker diabetic fatty rats: a difficult condition to reverse

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:

mark-yorek@uiowa.edu

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.

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)

untreated (9)

Enalapril (8)

ZDF lipoic

acid (8)

rosuvastatin (9)

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.

# 2007 The Authors

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

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.

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

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

xation

100Lean (16)ZDF untreated (11)ZDF enalapril (11)ZDF lipoic Acid (12)ZDF rosuvastatin (12)ZDF rosiglitazone (15)

†*††

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.

# 2007 The Authors

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

100Lean (15)ZDF untreated (14)ZDF enalapril (14)ZDF lipoic Acid (12)ZDF rosuvastatin (15)ZDF rosiglitazone (15)

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.

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.

# 2007 The Authors

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

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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