Progression of vascular and neural dysfunction in sciatic nerves of Zucker diabetic fatty and Zucker rats
Christine L. Oltman,
Lawrence J. Coppey,
Jill S. Gellett,
Eric P. Davidson,
Donald D. Lund, and
Mark A. Yorek
Veteran Affairs Medical Center and Department of Internal Medicine, University of Iowa, Iowa City, Iowa 52246
Submitted 16 December 2004
; accepted in final form 14 February 2005
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ABSTRACT
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We have examined the progression of vascular and neural deficits in Zucker rats, Zucker diabetic fatty (ZDF) diabetic rats, and age-matched lean ZDF rats from 8 to 40 wk of age. Both the ZDF diabetic and Zucker rats were glucose intolerant at 8 wk of age. The Zucker rats did not become hyperglycemic but were hyperinsulinemic through 32 wk of age. All ZDF diabetic rats became hyperglycemic by 8 wk of age. Through their life span, serum free fatty acids and triglycerides levels were significantly higher in Zucker and ZDF diabetic rats compared with age-matched lean ZDF rats. After 24 and 28 wk of age, endoneurial blood flow was significantly decreased in ZDF diabetic and Zucker rats. Motor nerve conduction velocity was significantly decreased after 1214 wk of age in ZDF diabetic rats and at 32 wk of age in Zucker rats. ACh-mediated vascular relaxation of epineurial arterioles of the sciatic nerve was impaired after 810 wk of age in ZDF diabetic rats and after
16 wk of age in Zucker rats. In contrast, vascular relaxation mediated by calcitonin gene-related peptide was impaired significantly after 28 wk of age in ZDF diabetic rats but not impaired in Zucker rats up to 40 wk of age. Markers of oxidative stress were differentially elevated in ZDF diabetic rats and Zucker rats. These data indicate that vascular and neural dysfunction develops in both Zucker and ZDF diabetic rats but at different rates, which may be the result of hyperglycemia.
diabetic neuropathy; vascular reactivity; oxidative stress; superoxide; type 2 diabetes; metabolic syndrome
THE PATHOGENESIS OF TYPE 2 human diabetes is characterized by obesity and many years of escalating insulin resistance, chronic hyperinsulinemia, and an ultimate failure of pancreatic islet
-cells to cope with the progressive demand for insulin (12, 39). The nature of this process means that type 2 diabetes often goes undiagnosed until the patient presents with chronic complications. Thus the sequential development of diabetic complications in type 2 diabetes is not well known because of the difficulty in identifying the precise onset of the disease. To address this issue, we have examined the progression of vascular and neural complications in diabetic fatty rats (ZDF/Drt-fa), an animal model for type 2 diabetes (27, 28, 34). In this obese diabetic rat model, all fatty males become hyperglycemic by 8 wk of age, and glucose remains elevated throughout their lifespan (27). Initially, Zucker diabetic fatty (ZDF) rats are hyperinsulinemic. However, by 2242 wk of age, serum insulin levels decline to below levels of insulin in age-matched lean control rats (27). A similar characteristic is seen in human type 2 diabetes, which is thought to be caused by pancreas/
-cell exhaustion. Through their life span, free fatty acids, triglycerides, and cholesterol levels are significantly higher in ZDF diabetic rats compared with lean littermate controls (27). In addition, ZDF diabetic rats develop neuropathy defined as a slowing of motor nerve conduction velocity (MNCV; see Refs. 9, 27, 28, 34).
For these studies, we have used age-matched lean littermates as controls. As an additional control, we have included the obese Zucker rat. The Zucker rat is the parent strain from which the ZDF diabetic rat was inbred. Therefore, the genetic backgrounds for these rats are very similar. The Zucker rat generally does not become hyperglycemic; however, it is insulin resistant, hypertensive, and dyslipidemic and has some degree of vascular dysfunction and thus is considered to be a good model for studies relating to the "metabolic syndrome" (2, 5, 19, 21, 42). Both the ZDF diabetic rat and the euglycemic Zucker rat have similar abnormalities in lipid metabolism; therefore, by comparing the outcome of these studies, we are able to begin to differentiate the impact of hyperglycemia and dyslipidemia on vascular and neural dysfunction.
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MATERIALS AND METHODS
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Unless stated otherwise, all chemicals used in these studies were obtained from Sigma Chemical (St. Louis, MO).
Animals.
Male Zucker, ZDF-lean (+/?), and ZDF-obese diabetic (fa/fa) rats 6 wk of age were obtained from Charles River Laboratories (Wilmington, MA). The animals were housed in a certified animal care facility, and food (Harlan Teklad no. 7001; Zucker and ZDF lean rats) or 7013 (ZDF) and water were provided ad libitum. All institutional (approval animal care and use review form no. 0290608) and National Institutes of Health guidelines for the use of animals were followed. After 840 wk of age, rats were used for studies described below. The studies were divided into nine time points based on the age of the rats (group 1: 810; group 2: 1214; group 3: 16; group 4: 20; group 5: 24; group 6: 28; group 7: 32; group 8: 36; and group 9: 40 wk of age).
On the day of the experiment, rats were anesthetized with Nembutal (50 mg/kg ip; Abbott Laboratories, North Chicago, IL), and nonfast blood glucose levels were determined with the use of glucose oxidase reagent strips (Lifescan, Milpitas, CA). Serum samples were collected for determination of insulin levels using a rat insulin RIA kit from LINCO Research (St. Charles, MO), and free fatty acid and triglyceride levels were determined using commercial kits from Roche Diagnostics (Mannheim, Germany) and Sigma Chemical, respectively. Afterward, MNCV and endoneurial blood flow (EBF) in the sciatic nerve were determined, and preparations were made for isolation of epineurial arterioles.
MNCV.
MNCV was determined as previously described using a noninvasive procedure in the sciatic-posterior tibial conducting system in a temperature-controlled environment (8, 9, 41). The MNCV was reported in meters per second. In these studies, we failed to perform measurements of sensory nerve conduction velocity (SNCV). Considering our results demonstrating that levels of calcitonin gene-related peptide (CGRP) are decreased in sensory nerves innervating epineurial arterioles of the sciatic nerve, it will be important for us to include determination of SNCV in future studies.
EBF.
Sciatic nerve EBF was determined as previously described using the hydrogen clearance method (8, 9, 41). The hydrogen clearance data were fitted to a mono- or biexponential curve using commercial software (Prism; GraphPad, San Diego, CA) and nutritive blood flow (ml·min1·100 g1), calculated using the equation described by Young (49), and vascular conductance (ml·min1·100 g1·mmHg1), determined by dividing nutritive blood flow by the average mean arterial blood pressure.
Vascular reactivity.
Videomicroscopy was used to investigate in vitro vasodilatory responsiveness of arterioles vascularizing the region of the sciatic nerve, as previously described (8, 9, 41). Cumulative concentration-response relationships were evaluated for CGRP (1011 to 108 M) and ACh (108 to 104 M) using vessels from each group of rats. At the end of each dose-response determination, a maximal dose of sodium nitroprusside (SNP, 104 M) was added. Afterward, papaverine (105 M) was added to determine maximal vasodilation, which was consistently the same as the vascular tone of the resting vessel at 40 mmHg.
Immunohistochemistry.
We analyzed for CGRP by immunohistochemical staining epineurial arterioles of the sciatic nerve from ZDF lean rats, Zucker rats, and ZDF diabetic rats at 28 and 40 wk of age. Epineurial arterioles of the sciatic nerve were collected as described above with minimal preparation. Afterward, the vessels were incubated for 30 min in buffer containing 1% Triton X-100 with 0.1% BSA (30, 47). The vessels were next washed with PBS and incubated in this buffer containing goat polyclonal antibodies against CGRP for 72 h at 4°C using the chambered cover glass system (Nalge Nunc, Naperville, IL). For controls, vessels were incubated in the absence of anti-CGRP (47). Afterward, vessels were washed with PBS and incubated in this buffer with Alexa-Fluor-555-conjugated donkey anti-goat IgG (Molecular Probes, Eugene, OR) for 24 h at 4°C. After incubation with the secondary antibody, vessels were washed with PBS and distilled water and then mounted with VectorShield. The labeled vessels derived from these studies were visualized with a Zeiss LSM 510 laser scanning confocal microscope using x40 objectives. Optical sections along the z-axis were collected at 1.0-µm intervals at a resolution of 512 x 512. Stacks of images were combined to form a single composite image.
Detection of superoxide and peroxynitrite.
Hydroethidine (Molecular Probes), an oxidative fluorescent dye, was used to evaluate in situ levels of superoxide in epineurial vessels as described previously (8, 9, 25). This method provides sensitive detection of superoxide in situ. Vessel segments from ZDF lean, Zucker, and ZDF diabetic rats after 28 and 40 wk of age were processed and imaged in parallel, and laser settings were identical for acquisition of all images. Superoxide levels were also measured using the aorta by lucigenin-enhanced chemiluminescence as described previously (25). Relative light units (RLU) were measured using a Zylux FB12 luminometer. Background activity was determined and subtracted, and RLU was normalized to surface area.
One of two mechanisms by which ACh can mediate vascular relaxation in arterioles that provide circulation to the sciatic nerve is through the production of nitric oxide (41). The chemistry of nitric oxide is complex, and several biochemical pathways other than nitric oxide production can influence nitric oxide action. For example, superoxide anion can interact with nitric oxide to form peroxynitrite (46). This reaction 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 cause cellular oxidative damage (4, 29). To determine whether formation of superoxide by arterioles that provide circulation to the sciatic nerve promotes the formation of peroxynitrite, we measured 3-nitrotyrosine (3-nitrotyrosine is a stable biomarker of tissue peroxynitrite formation) in vessel sections from 40-wk-old ZDF lean, Zucker, and ZDF diabetic rats. Briefly, frozen tissue segments of arterioles were cut into 5-µm sections and then incubated in PBS solution containing 1% Triton X-100 and 0.1% BSA for 30 min at room temperature. Afterward, the samples were incubated in this buffer solution containing mouse anti-nitrotyrosine (Upstate, Lake Placid, NY) overnight at 4°C. After being washed, the sections were incubated for 2 h with Alexa Fluor 555 goat anti-mouse IgG (Molecular Probes). Sections were then rinsed and mounted with VectorShield. The labeled vessels derived from these studies were visualized with a Zeiss LSM 510 laser scanning confocal microscope using x40 objectives.
Sciatic nerve glucose, sorbitol, and fructose content and Na+-K+-ATPase activity.
As a marker of metabolic derangement in the sciatic nerve, the level of glucose, sorbitol, and fructose and activity of Na+-K+-ATPase activity was determined. Intracellular content of glucose, sorbitol, and fructose was determined by gas-liquid chromatography, as previously described (8, 9, 41). Data were presented as nanomoles per milligram wet weight. Total and ouabain-inhibited Na+-K+-ATPase activities were measured in crude homogenates of sciatic nerve, as previously described (8, 41, 48). The activity was expressed as micromoles ADP per gram wet weight per hour.
Additional biological parameters.
Sciatic nerve-conjugated diene and serum thiobarbituric acid reactive substances (TBARS) levels were determined as additional markers of oxidative stress. TBARS level in serum was determined by the method of Mihara et al. (23) as modified by Siman and Eriksson (37). The data were reported as microgram per milliliter serum. Conjugated diene levels in sciatic nerve were determined by measuring the absorbance at 233 nm with extraction blanks used as references (8, 9). An extinction coefficient of 2.52 x 104 M was used to determine the amount of conjugated diene present. The data were reported as micrograms per milligram wet weight.
Data analysis.
The results are presented as means ± SE. Comparisons between the groups for body weight, blood glucose, MNCV, EBF, sciatic nerve Na+-K+-ATPase activity, sciatic nerve glucose, sorbitol and fructose content, sciatic nerve-conjugated diene levels, serum TBARS, serum free fatty acid, triglyceride and insulin levels, and lens glutathione levels were conducted using a one-way ANOVA and Newman-Keuls test for multiple comparisons (Prism software; GraphPad). Concentration-response curves for ACh- and CGRP-induced relaxation were compared using a two-way repeated-measures ANOVA with autoregressive covariance structure using proc mixed program of SAS (8, 9, 41). Whenever significant interactions were noted, specific treatment-dose effects were analyzed using a Bonferroni adjustment. A P value <0.05 was considered significant.
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RESULTS
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Body weight, blood glucose, and serum insulin levels.
Data in Fig. 1A demonstrate that Zucker rats continue to gain weight throughout most of the study period. In comparison, the weight of ZDF diabetic rats was initially greater than their lean littermate controls, but after 28 wk of age the lean ZDF rats were significantly heavier than the age-matched ZDF diabetic rats. The weight of ZDF diabetic rats began to decline after 28 wk of age, which corresponds to the time of significant depletion of serum insulin levels compared with age-matched lean ZDF rats (Fig. 1C). The blood glucose level was significantly increased in ZDF diabetic rats compared with age-matched lean ZDF rats at all time points of the study (Fig. 1B). The blood glucose level in Zucker rats was similar to lean ZDF rats for all age groups between 8 and 36 wk of age but increased significantly after 40 wk of age (Fig. 1B), which corresponds to the time of a decrease in serum insulin levels (Fig. 1C). At 8 wk of age, we performed glucose tolerance test on four rats from each of the three groups according to Farrar et al. (16). At 8 wk of age, the ZDF diabetic and Zucker rats were found to be glucose intolerant compared with lean ZDF rats (data not shown). This is consistent with previous studies (16, 31). Mean arterial blood pressure was also determined. At 814 wk of age (groups 1 and 2) and 3640 wk of age (groups 8 and 9), the mean arterial blood pressure for lean ZDF rats, ZDF diabetic rats, and Zucker rats was 120 ± 4 and 117 ± 6, 129 ± 4 and 146 ± 5, and 129 ± 3 and 139 ± 4 mmHg, respectively.

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Fig. 1. Determination of change in weight, blood glucose, and serum insulin in lean Zucker diabetic fatty (ZDF; lean), ZDF diabetic (ZDF), and Zucker rats from 8 to 40 wk of age. Changes in weight (A), blood glucose levels (B), and serum insulin levels (C) were determined in 9 groups of rats. The groups (G1-G9) are based on age (G1: 810; G2: 1214; G3: 16; G4: 20; G5 24; G6: 28; G7: 32; G8: 36; and G9: 40 wk of age). Data are presented as means ± SE. *P < 0.05 compared with age-matched lean ZDF rats. Data were derived from 69 lean ZDF, ZDF diabetic, or Zucker rats in each of the groups.
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Serum triglyceride and free fatty acid levels.
Serum triglyceride and free fatty acid levels were significantly increased throughout the study period (840 wk of age) in both ZDF diabetic and Zucker rats (Fig. 2).

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Fig. 2. Determination of change in serum triglycerides (TG) and free fatty acids (FFA) in lean ZDF, ZDF diabetic, and Zucker rats from 8 to 40 wk of age. The details for each group of rats regarding the age of the rats and no. of animals in each group are the same as described in the legend for Fig. 1. Data are presented as means ± SE. *P < 0.05 compared with age-matched lean ZDF rats.
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MNCV and EBF.
Data in Fig. 3 demonstrate that MNCV was significantly reduced after 1214 wk of age and 32 wk of age in ZDF diabetic and Zucker rats, respectively, compared with age-matched lean ZDF rats. EBF in the sciatic nerve was significantly decreased after 24 wk of age and 2832 wk of age in ZDF diabetic and Zucker rats, respectively, compared with age-matched lean ZDF rats (Fig. 4).

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Fig. 3. Determination of motor nerve conduction velocity (MNCV) in lean ZDF, ZDF diabetic, and Zucker rats from 8 to 40 wk of age. The details for each group of rats regarding the age of the rats and no. of animals in each group are the same as described in the legend for Fig. 1. Data are presented as means ± SE. *P < 0.05 compared with age-matched lean ZDF rats.
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Fig. 4. Determination of endoneurial blood flow (nutritive and conductance) in the sciatic nerve in lean ZDF, ZDF diabetic, and Zucker rats from 8 to 40 wk of age. The details for each group of rats regarding the age of the rats and no. of animals in each group are the same as described in the legend for Fig. 1. Data are presented as means ± SE. *P < 0.05 compared with age-matched lean ZDF rats.
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Evaluation of oxidative stress.
We have previously reported that diabetes causes an increase in superoxide and peroxynitrite levels in epineurial arterioles that provide circulation to the sciatic nerve from streptozotocin-induced diabetic rats of 34 wk duration (8, 9). In these studies, we sought to determine whether superoxide and peroxynitrite levels are changed in Zucker rats and ZDF diabetic rats compared with normoglycemic lean ZDF rats. Furthermore, to assess the effect of the metabolic syndrome and diabetes on oxidative stress, we examined three additional markers of oxidative stress in several different tissues. By measuring different markers of oxidative stress in different tissues, we were able to get a more complete understanding of the oxidative stress status of the rats used in these studies. Data in Fig. 5 demonstrate that superoxide level was increased in epineurial arterioles of the sciatic nerve from ZDF diabetic rats after 28 and 40 wk of age compared with age-matched lean ZDF rats. In contrast, superoxide level appeared to be increased in epineurial arterioles from Zucker rats after 28 wk of age but not after 40 wk of age. We also measured superoxide levels in the aorta using lucigenin-enhanced chemiluminescence (25). At 28 and 40 wk of age, superoxide level was increased in the aorta of Zucker rats and to a lesser extent in ZDF diabetic rats compared with age-matched lean ZDF rats (3.28 ± 0.16 and 3.33 ± 0.14, 5.98 ± 0.69 and 7.59 ± 0.95, and 4.55 ± 0.54 and 6.33 ± 0.91 RLU for ZDF lean rats, Zucker rats, and ZDF diabetic rats after 28 and 40 wk of age, respectively, n = 6). Data in Fig. 6 demonstrate that peroxynitrite level was increased in epineurial arterioles from 40-wk-old ZDF diabetic rats compared with age-matched lean ZDF rats. In contrast, peroxynitrite level was not increased in epineurial arterioles from 40-wk-old Zucker rats. Serum TBARS level was also measured and was found to be moderately increased in ZDF diabetic and Zucker rats after 28 and 40 wk of age (0.48 ± 0.01 and 0.33 ± 0.02, 0.60 ± 0.04 and 0.43 ± 0.01, and 0.70 ± 0.05 and 0.40 ± 0.03 µg/ml serum for lean ZDF rats, ZDF diabetic rats, and Zucker rats after 28 and 40 wk of age, respectively). Sciatic nerve-conjugated diene level was also modestly increased in ZDF diabetic rats and Zucker rats (2.6 ± 0.6 and 1.6 ± 0.8, 5.1 ± 0.6 and 5.6 ± 0.7, and 4.3 ± 0.8 and 4.3 ± 0.5 µmol/mg wet wt for lean ZDF rats, ZDF diabetic rats, and Zucker rats after 28 and 40 wk of age, respectively). We also examined the sciatic nerve for sorbitol and fructose level and activity of Na+-K+-ATPase. Sorbitol and fructose levels were increased in the sciatic nerve of ZDF diabetic rats compared with lean ZDF rats and unchanged in sciatic nerves from Zucker rats after 40 wk of age (1.4 ± 0.1 and 0.6 ± 0.1, 2.5 ± 0.5 and 2.5 ± 0.3, and 1.7 ± 0.4 and 1.4 ± 0.2 nmol/mg wet wt for sorbitol and fructose for lean ZDF rats, ZDF diabetic rats, and Zucker rats after 40 wk of age, respectively). The glucose level in the sciatic nerve was also significantly increased in ZDF diabetic rats at 40 wk of age compared with lean ZDF rats and Zucker rats (8.8 ± 1.1, 9.9 ± 0.9, and 31.7 ± 3.8 nmol/mg wet wt for lean ZDF rats, Zucker rats, and ZDF diabetic rats, after 40 wk of age, respectively). Similar results were obtained from rats that were 28 wk old. Sciatic nerve Na+-K+-ATPase activity was not changed in lean ZDF rats, ZDF diabetic rats, or Zucker rats (data not shown).

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Fig. 5. Detection of superoxide in epineurial arterioles of the sciatic nerve from lean ZDF, Zucker, and ZDF diabetic rats at 28 or 40 wk of age. Presented are fluorescent photomicrographs of confocal microscopic sections of epineurial arterioles of the sciatic nerve from lean ZDF, Zucker and ZDF diabetic rats. Each vessel was examined on the same day. Arterioles were labeled with the oxidative dye hydroethidine as described in MATERIALS AND METHODS. Recording of fluorescence was taken at identical laser and photomultiplier settings for each vessel cross section. Shown is a representative sample from multiple sections from each evaluation.
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Fig. 6. Detection of peroxynitrite in epineurial arterioles of the sciatic nerve from lean ZDF, Zucker, and ZDF diabetic rats at 40 wk of age. Presented are fluorescent photomicrographs of confocal microscopic sections of epineurial arterioles of the sciatic nerve from lean ZDF, Zucker, and ZDF diabetic rats. Each vessel was examined on the same day. Arterioles were labeled as described in MATERIALS AND METHODS. Recording of fluorescence was taken at identical laser and photomultiplier settings for each vessel cross section. Shown is a representative sample from multiple sections from each evaluation.
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Immunohistochemical staining for CGRP in epineurial arterioles of the sciatic nerve.
To examine CGRP level in sensory neurons innervating epineurial arterioles of the sciatic nerve, we conducted immunohistochemical staining studies using arterioles from lean ZDF rats, ZDF diabetic rats, and Zucker rats after 40 wk of age. Previously, we demonstrated that nerve fascicles innervating epineurial arterioles of the sciatic nerve are immunoreactive for CGRP and that streptozotocin-induced diabetes, 1012 wk duration, caused a decrease in the CGRP content in epineurial arterioles (47). Data in Fig. 7 illustrate that the CGRP level was severely decreased in epineurial arterioles from ZDF diabetic rats and moderately decreased in epineurial arterioles from Zucker rats compared with epineurial arterioles from lean ZDF rats at 40 wk of age. No staining was observed in any of these vessels when the primary antibody was absent in the incubation (data not shown; see Ref. 47).

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Fig. 7. Detection of calcitonin gene-related peptide (CGRP) in epineurial arterioles of the sciatic nerve from lean ZDF, Zucker, and ZDF diabetic rats at 40 wk of age. Presented are fluorescent photomicrographs of confocal microscopic images of epineurial arterioles of the sciatic nerve from lean ZDF, Zucker, and ZDF diabetic rats. The images were created by combining Z-sections after scanning the entire vessel. The vessels were examined on the same day. Arterioles were labeled as described in MATERIALS AND METHODS. Recording of fluorescence was taken at identical laser and photomultiplier settings for each vessel section.
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Arteriolar vascular reactivity.
Previously, we demonstrated that exogenous ACh (endothelium dependent) and CGRP (endothelium independent) cause a concentration-dependent relaxation of epineurial arterioles from the sciatic nerve (41, 47). In these studies, changes in vascular diameter of epineurial arterioles of the sciatic nerve were measured in vitro by application of ACh and CGRP. Baseline diameter of vessels from lean ZDF rats, ZDF diabetic rats, and Zucker rats was similar, and the vessels were preconstricted to a similar degree with phenylephrine (106 M). Data in Fig. 8 demonstrate that ACh-mediated vasodilation was significantly impaired in epineurial arterioles from ZDF diabetic rats compared with lean ZDF rats after 810 wk of age and more severely impaired after 40 wk of age. ACh-mediated vasodilation was normal in epineurial arterioles from Zucker rats after 810 wk of age but severely impaired after 40 wk of age. Data in Fig. 9 demonstrate that CGRP-mediated relaxation was normal after 810 wk of age in both ZDF diabetic rats and Zucker rats but impaired at suboptimal doses of CGRP after 40 wk of age in Zucker rats and to a greater extent in ZDF diabetic rats. We also examined the affect of age on vascular relaxation mediated by suboptimal concentrations of ACh (105 M) and CGRP (109 M) and a maximal dosage of SNP (104 M) in epineurial arterioles of the sciatic nerve. Data in Fig. 10A demonstrate that ACh-mediated vasodilation was significantly impaired at all ages of ZDF diabetic rats and after 16 wk of age in Zucker rats. Vascular relaxation by epineurial arterioles mediated by 109 M CGRP was impaired in ZDF diabetic rats after 28 wk of age but was not impaired at this dose of CGRP in Zucker rats (Fig. 10B). Vascular relaxation in response to a maximum dose of SNP was significantly impaired in ZDF diabetic rats after 24 wk of age (Fig. 10C). SNP relaxation also seemed to be impaired in epineurial arterioles from Zucker rats, but significance was only realized in the 24- and 40-wk-old groups. Age does not appear to influence vascular relaxation mediated by ACh, CGRP, or SNP in lean ZDF rats (Fig. 10, AC).

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Fig. 8. Determination of ACh-mediated vascular relaxation in epineurial arterioles of the sciatic nerve from lean ZDF, ZDF diabetic, and Zucker rats after 810 (G1) or 40 (G9) wk of age. Pressurized arterioles (40 mmHg) were constricted with phenylephrine (3050%), and incremental doses of ACh were added to the bathing solution while recording steady-state vessel diameter. Data are presented as the mean of %relaxation ± SE. *P < 0.05 compared with age-matched lean ZDF rats.
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Fig. 9. Determination of CGRP-mediated vascular relaxation in epineurial arterioles of the sciatic nerve from lean ZDF, ZDF diabetic, and Zucker rats after 810 (G1) or 40 (G9) wk of age. Pressurized arterioles (40 mmHg) were constricted with phenylephrine (3050%), and incremental doses of CGRP were added to the bathing solution while recording steady state-vessel diameter. Data are presented as the mean of %relaxation ± SE. *P < 0.05 compared with age-matched lean ZDF rats.
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Fig. 10. Determination of vascular relaxation mediated by 105 M ACh (A), 109 M CGRP (B), and 104 M sodium nitroprusside (SNP, C) in epineurial arterioles of the sciatic nerve from lean ZDF, ZDF diabetic, and Zucker (Zuck) rats after 840 wk of age. Pressurized arterioles (40 mmHg) were constricted with phenylephrine (3050%), and incremental doses of ACh, CGRP, or SNP were added to the bathing solution while recording steady-state vessel diameter. The data presented are the amount of relaxation recorded for 105 M ACh, 109 M CGRP, and 104 M SNP. The details for each group of rats regarding the age of the rats and no. of animals in each group are the same as described in the legend for Fig. 1. Data are presented as the mean of %relaxation ± SE. *P < 0.05 compared with age-matched lean ZDF rats.
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DISCUSSION
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Previously we demonstrated that, in streptozotocin-induced diabetic rats, a model for type I diabetes, impairment of vascular function and reduction in EBF precedes slowing of MNCV (8). In the present study, we were interested in determining the sequential development of vascular and neural dysfunction in rat models of type 2 diabetes and the metabolic syndrome. The key findings of these studies were, in Zucker rats, the steady decline with age in vascular reactivity to ACh in epineurial arterioles of the sciatic nerve, the slowing in MNCV, and reduction of EBF of the sciatic nerve. In ZDF diabetic rats, the key findings were the early impairment and extensive deterioration in vascular reactivity to ACh and the impaired response to CGRP and decrease in CGRP content in epineurial arterioles of the sciatic nerve.
In Zucker rats, the progression of vascular dysfunction in response to ACh in epineurial arterioles of the sciatic nerve and reduction in MNCV and EBF in the sciatic nerve occurred independent of hyperglycemia. To the best of our knowledge, this is the first time that it has been shown that reduction in MNCV can occur in normoglycemic animals that have characteristics consistent with the metabolic syndrome. If these results are translational to humans, it would suggest that obese patients with dyslipidemia and hypertension may be susceptible to peripheral nerve dysfunction and damage.
In ZDF diabetic rats, early after the onset of hyperglycemia there was a steady and severe deterioration in vascular reactivity to ACh. After 24 wk of age, vasodilation in response to SNP in ZDF diabetic rats was also impaired. In Zucker rats, there was a tendency for an impairment in vasodilation mediated by SNP, but it was only found to be significant at two time points and likely only physiologically relevant after 40 wk of age. These data suggest that the response by the smooth muscle cells to a nitric oxide donor in epineurial arteriole vasculature diminishes with age, and the rate of decline is likely influenced by hyperglycemia.
Previously, we have demonstrated that vascular relaxation to ACh in epineurial arterioles of the sciatic nerve is mediated by the following two mechanisms: formation of nitric oxide and endothelium-derived hyperpolarizing factor (EDHF) (41). It is likely that the impairment in vascular function we observed in epineurial arterioles in response to ACh in ZDF diabetic rats was because of reduced activity of both mechanisms. Superoxide levels are increased in the epineurial arterioles of ZDF diabetic rats, and at 40 wk of age we also observed an increase in peroxynitrite levels in these vessels. This suggests that quenching of nitric oxide through formation of peroxynitrite may be diminishing the availability of nitric oxide for vasodilation (9, 46). However, in Zucker rats, the reason for the decrease in ACh-mediated relaxation in epineurial arterioles may be different than in ZDF diabetic rats. The increase in superoxide in epineurial arterioles from Zucker rats was inconsistent and less than was observed in ZDF diabetic rats. Moreover, after 40 wk of age, we did not observe an increase in peroxynitrite staining in epineurial arterioles from Zucker rats compared with age-matched lean animals. This suggests that nitric oxide availability for vasodilation was not quenched by superoxide. Therefore, it seems likely that reduced vasodilation to ACh in epineurial arterioles from Zucker rats may be the result of reduced EDHF formation or bioactivity (45). Other studies have indicated that impaired vasodilation in cerebral and coronary arteries from Zucker rats and fructose-fed rats is the result of calcium-dependent potassium channel dysfunction (14, 15, 24). Further studies will be necessary to confirm these results and determine the mechanism(s) responsible for impaired vasodilation in epineurial arterioles from Zucker rats.
We found that vascular relaxation in response to exogenous CGRP was impaired in epineurial arterioles from ZDF diabetic rats after 2840 wk of age but not impaired in epineurial arterioles from Zucker rats. CGRP levels in sensory nerves innervating epineurial arterioles of the sciatic nerve were also decreased in ZDF diabetic rats and to a lesser degree in Zucker rats. The results from the CGRP studies in ZDF diabetic rats were similar to the results from our studies with streptozotocin-induced diabetic rats (47). Because exogenous CGRP-mediated relaxation of epineurial arterioles of the sciatic nerve from Zucker rats was not impaired, it suggests that hyperglycemia is a contributing factor to decreased CGRP-mediated relaxation. The decrease in exogenous CGRP-mediated vasodilation could be the result of impaired signaling mechanism(s) or a decrease in binding for CGRP to its receptor on smooth muscle cells. Functional CGRP receptors consist of three different proteins: the calcitonin receptor-like receptor, receptor activity-modifying proteins, and the receptor component protein (17, 22, 33). If the expression of one or more of these proteins is compromised by diabetes/hyperglycemia, the function of the CGRP receptor would likely be affected. The vasodilatory response to CGRP involves the formation of cAMP and activation of Ca2+ signaling pathways (1, 7, 18, 35). It is also possible that the signaling mechanism(s) activated by the CGRP receptor may be impaired in diabetes/hyperglycemia, thereby causing a decrease in vasodilation in response to exogenous CGRP.
The decrease in CGRP levels in sensory nerves innervating epineurial arterioles of ZDF diabetic rats could be because of a decrease in axonal transport of CGRP from the dorsal root ganglion cells or denervation. In the sciatic nerve, Tomlinson and colleagues reported that diabetes caused deficits in both anterograde and retrograde axonal transport of CGRP and a 3040% decrease in CGRP content, which was prevented by insulin or treatment with nerve growth factor (13, 43, 44). The reason for decreased CGRP bioactivity and CGRP levels in sensory nerves innervating epineurial arterioles of the sciatic nerve in diabetes is presently under investigation. Impairment of CGRP levels and bioactivity could have an impact on the development and progression of diabetic neuropathy since the neuropeptides released by sensory nerves are thought to regulate vascular tone and blood flow to peripheral nerves (3, 20, 32, 50). Compromising the level and function of these neuropeptides could contribute to nerve ischemia and morphological changes observed during the progression of diabetic neuropathy.
Changes in vascular reactivity to ACh in epineurial arterioles in Zucker and ZDF diabetic rats preceded the reduction of EBF and slowing of MNCV in the sciatic nerve. With one exception, the data on vascular function of epineurial arterioles and MNCV in ZDF diabetic rats are similar to the results we obtained with streptozotocin-induced diabetic rats (8). The exception was that the slowing of MNCV preceded the decrease in EBF. The reason for this is unknown but suggests that impaired vascular function of epineurial arterioles may be a better indicator of developing vascular and neural damage than changes in EBF.
The data demonstrating that EBF and MNCV are decreased in Zucker rats are novel and important. Unlike the data from ZDF diabetic rats, the decrease in EBF in Zucker rats preceded the decrease in MNCV, suggesting that the two events may be related and that the reduction in EBF may be a factor in nerve functional changes. The etiology of the changes in EBF and MNCV in the sciatic nerve of Zucker rats will require further investigation. However, it seems likely that neural dysfunction in Zucker rats will be linked to impaired vascular function, since reduced vasodilation to ACh is an early event in this model. As mentioned above, it seems unlikely that reduced ACh-mediated vasodilation in epineurial arterioles from Zucker rats was the result of formation of superoxide and quenching of nitric oxide, since we were not able to detect increased superoxide or peroxynitrite formation in epineurial arterioles of Zucker rats at 40 wk of age. However, increased oxidative stress may contribute to vascular dysfunction in other vessels in Zucker rats, since we were able to demonstrate increased superoxide formation in the aorta compared with age-matched lean control rats after 28 and 40 wk of age.
In ZDF diabetic rats, the level of superoxide in epineurial arterioles and aorta was greater at 40 wk of age than at 28 wk of age. In Zucker rats, the level of superoxide was increased from 28 to 40 wk of age in the aorta but was decreased in epineurial arterioles from 28 to 40 wk of age. Part of the reason for these differences is likely due to different sources of superoxide production between the aorta and epineurial arterioles of the sciatic nerve. In the aorta, the major source of superoxide formation is probably NAD(P)H oxidase (40). In epineurial arterioles of the sciatic nerve, we have previously demonstrated that the mitochondria are likely the major source of superoxide formation in diabetes (11). Increased superoxide formation by the mitochondria is dependent on hyperglycemia and blocked by uncoupling of oxidative phosphorylation (26). Because Zucker rats were not hyperglycemic until 40 wk of age, and even then the level of hyperglycemia was less than ZDF diabetic rats, it is not surprising that superoxide levels were not consistently increased in epineurial arterioles. Other factors may also be influencing the difference in superoxide formation in epineurial arterioles of the sciatic nerve of ZDF diabetic and Zucker rats. Expression of antioxidant defense mechanisms is likely different in the two models. For instance, we know that the levels of glutathione in the lens and sciatic nerve remain normal in Zucker rats up to 40 wk of age but are decreased significantly at 812 wk of age in ZDF diabetic rats (data not shown). The same may be true for other antioxidant defense mechanisms. We also observed a difference in the presence of superoxide in epineurial arterioles of 28- and 40-wk-old Zucker rats. We do not understand the influence insulin resistance and hyperinsulinemia has on superoxide formation. Under insulin-resistant conditions, levels of tetrahydrobiopterin are reduced, and the production of superoxide by nitric oxide synthases is increased (36). In our studies, serum levels of insulin were increased at 28 wk of age, but, after 3640 wk of age, serum insulin levels were decreased to near control values. It is interesting to speculate that hyperinsulinemia and hyperglycemia may influence superoxide formation. These issues will require further investigation.
In summary, these studies have demonstrated that impairment of vascular relaxation to ACh in epineurial arterioles of the sciatic nerve occurs early after the onset of hyperglycemia in ZDF diabetic rats and after
16 wk of age in Zucker rats. Surprisingly, in Zucker rats, we observed a decrease in EBF and MNCV in the sciatic nerve. This could have important implications if similar changes occur in human patients. Superoxide and peroxynitrite formation were increased in epineurial arterioles of the sciatic nerve from ZDF diabetic rats but not Zucker rats after 40 wk of age. This suggests that different mechanisms may be contributing to impairment of vascular function in epineurial arterioles of the sciatic nerve in ZDF diabetic and Zucker rats. In addition, we observed that vascular reactivity to CGRP was decreased in epineurial arterioles from ZDF diabetic rats but not Zucker rats. However, innervation of epineurial arterioles by sensory nerves containing CGRP was decreased in Zucker rats and to a greater extent in ZDF diabetic rats. Progressive changes in both peripheral vascular and neural function occurred in ZDF diabetic and normoglycemic/obese Zucker rats. Some of the differences in the development and progression of these changes were likely the result of hyperglycemia. In ZDF diabetic rats, there is a chronic condition of hyperglycemia that likely contributes to vascular and neural dysfunction. However, in Zucker rats, hyperglycemia was not apparent until 40 wk of age, well after the onset of vascular and neural dysfunction. In humans, impaired glucose tolerance serves as a marker of insulin resistance and is a predictor for both large- and small-vessel vascular complications (38). Furthermore, it has been shown that postprandial hypertriglyceridemia and hyperglycemia have independent and cumulative effects on endothelial function (6). Zucker rats demonstrate glucose intolerance as early as 8 wk of age, and whether this contributes to vascular dysfunction over time in epineurial arterioles of the sciatic nerve needs to be considered.
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GRANTS
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This work was supported by a Veterans Affairs Merit Review.
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FOOTNOTES
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Address for reprint requests and other correspondence: M. A. Yorek, 3 E 17 Veteran Affairs Medical Center, Iowa City, IA 52246 (e-mail: mark-yorek{at}uiowa.edu)
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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