Indices of cardiovascular autonomic neuropathy (CAN) in experimental models of Type 1 diabetes mellitus (T1DM) are often contrary to clinical data. Here, we investigated whether a relatable insulin-treated model of T1DM would induce deficits in cardiovascular (CV) autonomic function more reflective of clinical results and if exercise training could prevent those deficits. Sixty-four rats were divided into four groups: sedentary control (C), sedentary T1DM (D), control exercise (CX), or T1DM exercise (DX). Diabetes was induced via multiple low-dose injections of streptozotocin and blood glucose was maintained at moderate hyperglycemia (9–17 mM) through insulin supplementation. Exercise training consisted of daily treadmill running for 10 weeks. Compared to C, D had blunted baroreflex sensitivity, increased vascular sympathetic tone, increased serum neuropeptide Y (NPY), and decreased intrinsic heart rate. In contrast, DX differed from D in all measures of CAN (except NPY), including heart rate variability. These findings demonstrate that this T1DM model elicits deficits and exercise-mediated improvements to CV autonomic function which are reflective of clinical T1DM.
A common and serious complication of Type 1 diabetes mellitus (T1DM) is diabetic autonomic neuropathy [
Exercise has been demonstrated to be an effective means of improving deficits in HRV and BRS in both clinical and experimental T1DM [
Another important difference between experimental and clinical T1DM is the common omission of insulin treatment in experimental diabetes leading to severe hyperglycemia ranging from roughly 17 to 25 mM blood glucose concentrations ([BG]) [
Yet, despite the use of insulin therapy in clinical T1DM, it is often the case that chronic, moderate hyperglycemia is maintained as a result of difficulties in regulating [BG] in response to dynamic influences on glycemic control, such as food intake and exercise [
The purpose of the current study was to investigate whether our model of multiple low-dose STZ-induced T1DM with insulin therapy would induce deficits in cardiovascular autonomic function more representative of clinical T1DM, and if high intensity aerobic training could prevent those deficits. We hypothesised that (1) our model of STZ-induced T1DM would elicit indices of CAN, including a blunted BRS (bradycardia and tachycardia response), lowered HRV and intrinsic heart rate, increased vascular sympathetic tone, and increased mean arterial pressure, and (2) high intensity aerobic exercise training would prevent or ameliorate the indications of CAN.
The protocols used in this investigation were approved by the University of Western Ontario Council on Animal Care and conformed to the guidelines of the Canadian Council on Animal Care.
Eight-week-old male Sprague-Dawley rats were obtained from Charles River Laboratories Canada (Saint-Constant, Quebec). The rats were housed in pairs and maintained on a 12-hour dark/light cycle at a constant temperature (
Sixty-four rats were randomly assigned to one of four groups as follows: (1) sedentary control (C,
Upon arrival rats were acclimatized to the laboratory setting for five days. Subsequently, T1DM was induced over five consecutive days by multiple intraperitoneal (IP) injections of 20 mg/kg streptozotocin (STZ, Sigma-Aldrich) dissolved in a citrate buffer (0.1 M, pH 4.5). Diabetes was confirmed by blood glucose measurements greater than or equal to 18 mM on two consecutive days. If necessary, subsequent 20 mg/kg STZ injections were administered until diabetes was confirmed. Following the confirmation of diabetes, insulin pellets (1 pellet; 2 U insulin/day; Linplant, Linshin Canada, Inc., Toronto, Ontario, Canada) were implanted subcutaneously in the abdominal region. Insulin pellet doses were then monitored for 1 week and adjusted (±0.5 pellets) in order to obtain daily nonfasting blood glucose concentrations in the moderate hyperglycemic range of 9–17 mM. Insulin dose was determined by multiplying the total quantity of pellet implanted (0.5 pellet increments) by the amount of insulin released per pellet (2 units of insulin/day/pellet) divided by the body weight (Kg) of the rat.
Body weights and nonfasting blood glucose concentrations were obtained weekly. Blood was obtained from the saphenous vein by venous puncture with a 30-gauge needle and measured via Freestyle Lite Blood Glucose Monitoring System (Abbott Diabetes Care Inc., Mississauga, Ontario, Canada).
Intravenous glucose tolerance tests (IVGTT) were performed on all animals prior to T1DM induction (pre-T1DM) and at the end of week 10 of the exercise training period. Rats were fasted for approximately 8 to 12 hours prior to the assay and did not perform exercise on the day of their IVGTT. A sterile-filtered dextrose solution (50% dextrose, 50% ddH2O) was injected (1 g/kg) into the lateral tail vein of the conscious rat. Following dextrose infusion, blood glucose was measured at 5 minutes, at 10 minutes, and then at 10-minute intervals thereafter until blood glucose levels plateaued.
Prior to the initiation of the exercise training program, rats were familiarized with the exercise equipment on two consecutive days. The familiarization consisted of two 15-minute sessions of running at progressive treadmill speeds up to 30 meters per minute (m/min). The treadmill was a custom-built apparatus fabricated by the physical plant at University of Western Ontario and has been used in many previous studies [
To achieve a surgical plane of anesthesia, rats were placed in an induction chamber circulating 4% isoflurane (96% O2). Once motor reflexes were undetectable, rats were transferred to a nosecone delivering 3% isoflurane (97% O2) and placed on a hot water pad (37°C). Rats were cannulated with saline-infused polyethylene (PE90) catheters in the right jugular vein and carotid artery and each catheter was attached to a three-way stopcock. The jugular vein catheter was used for drug infusions and the carotid artery catheter was connected in series with a pressure transducer (PX272, Edwards Life Sciences, Irvine, California, USA) for arterial blood pressure measurements.
At the end of the preparative surgery, rats were injected IP with a 25 mg/Kg “cocktail” of urethane (16 mg/mL) and
Heart rate (HR), systolic blood pressure (SBP), and mean arterial pressure (MAP) were determined from the blood pressure pulse waveform and were collected while the rats were under urethane/
Prior to drug infusions, 5 minutes of spontaneous electrocardiogram data was sampled at 1000 Hz and analyzed with LabChart HRV analysis software (ADInstruments). Time domain analysis of the standard deviation between normal peak pulses of the pressure pulse waveform (SDNN) was quantified as a measure of the total variability of the HR. Frequency domain analysis of the high frequency (HF) band of the Fast Fourier Transform (FFT) of the data was assessed as an index of parasympathetically mediated HRV.
Baroreflex sensitivity (BRS) was assessed using the modified Oxford technique [
To measure the sympathetic contribution to baseline vascular resistance, MAP was assessed before and after a bolus injection of the
To ensure physiological testing did not confound serum neuropeptide Y concentration [NPY] measurement, a subset of animals from each group did not undergo surgery for heart rate variability, baroreflex sensitivity, mean arterial pressure, or vascular sympathetic tone measurements. Rather, at the end of the 10-week exercise training period these animals were anesthetized via intraperitoneal injection of sodium pentobarbital (65 mg/kg) and blood serum samples for [NPY] measurement were collected upon euthanasia. Serum [NPY] was measured using an NPY ELISA kit (USCN Life Sciences Inc.) according to the manufacturer’s instructions.
A Langendorff preparation was used to measure intrinsic heart rate. Following the euthanasia of animals for blood [NPY] measurement, hearts were extracted and immediately arrested by placing them in ice cold Krebs-Henseleit buffer (KHB). Hearts were cannulated for unpaced retrograde aortic constant flow perfusion (15 mL/min) of coronary arteries with KHB (containing 120 mM NaCl, 4.63 M KCl, 1.17 mM KH2PO4, 1.25 mM CaCl2, 1.2 mM MgCl2, 20 mM NaHCO3, and 8 mM glucose gassed with 95% O2 and 5% CO2) that was maintained at 37°C [
Body weight and blood glucose concentrations were compared using a two-way repeated measures ANOVA, while endpoint measures were compared by two-way ANOVA, with the exception of endpoint insulin dose, which was compared using a one-tailed
All groups increased in body weight over the course of the study (
(a) Weekly body weights: C, sedentary control (
The glucose clearance rate (
(a) IVGTT glucose clearance rate (
For resting HR and MAP, there was not a significant difference between groups at week 10 (Figures
(a) Heart rate (beats per minute) and (b) mean arterial pressure at week 10. C, sedentary control (
Total HRV at week 10, as measured by the standard deviation of the normal pulse wave peaks (SDNN), was not significantly different between groups (Figure
(a) Total HRV (SDNN) at week 10: C, sedentary control (
In response to SNP infusion, there was not a significant difference between groups in the tachycardia BRS response (Figure
(a) Tachycardia baroreflex response sensitivity to sodium nitroprusside at week 10: C, sedentary control (
An interaction between T1DM and exercise was observed for the prazosin-induced change in MAP (
(a) Vascular sympathetic tone (VST) at week 10. This was determined by measuring the percent change in MAP after prazosin treatment at week 10: C, sedentary control (
This study demonstrated that a multiple low-dose STZ model with moderate hyperglycemia, maintained using insulin therapy, produced deficits in cardiovascular autonomic function without inducing the resting bradycardia or hypotension typical of other STZ models. This study also showed that high intensity aerobic exercise training can prevent deficits of cardiovascular autonomic function caused by T1DM. Furthermore, because [BG] was held within a moderate hyperglycemic range, the observed exercise-mediated improvements to indications of CAN were independent of changes in [BG] and, instead, may primarily have been the result of improvements to other aspects of glucoregulation and/or the preservation of autonomic nervous system function.
Although we found time domain analysis of total HRV, as measured by the SDNN, did not demonstrate differences between groups, frequency domain analysis exposed a reduction in the HF power in the D group compared with the DX group. Since the HF power corresponds to the level of vagally mediated parasympathetic HRV, these results demonstrate not only the detrimental effects of T1DM on autonomic cardiac control but also the benefits of exercise training toward ameliorating those effects. These findings are similar to those of other experiments of both experimental [
Both tachycardia and bradycardia responses were studied in the context of BRS analysis in order to explore the control features related to unloading or loading of the baroreceptors, respectively. Some discrepancy exists between different experimental models of T1DM and their impact on BRS measures. Investigations using the hyperglycemic Non-Obese Diabetic (NOD) T1DM mouse model have shown elevations in BRS measures rather than attenuated responses [
However, the current study provides evidence that the ability of exercise to ameliorate cardiovascular autonomic dysfunction may be independent of its ability to reduce [BG], which challenges the direct relationship between [BG] and CAN suggested by previous studies [
An alternative mechanism by which exercise can influence BRS was reported by Bernardi et al. (2011), who elucidated the importance of tissue oxygenation in T1DM [
Another interesting outcome of the current study was the alteration of sympathetic vasomotor control in the D group, which was also modified by concurrent exercise training. In this study, prazosin treatment resulted in a drop in MAP that was approximately twofold greater in the D group compared to the C and DX groups, which is indicative of a much greater sympathetic contribution to the maintenance of baseline vascular resistance [
The conclusion above regarding sympathetic hyperactivity in the D group is supported by measurements of neuropeptide Y [NPY] obtained in this study. [NPY] is coreleased with norepinephrine from perivascular and cardiac sympathetic nerve terminals during sympathetic activation [
Despite improvements by exercise training to deficits of cardiovascular autonomic function, no observable statistical differences in either MAP or HR were evident between any of the groups. Indeed, it has been shown that alterations in autonomic function occur before or without alterations in MAP and HR and are uncorrelated to changes in sympathetic tone [
To evaluate the heart rate of these animals without neural influence, we measured the beat rate of denervated hearts using the isolated Langendorff technique. We found that the IHR of the D group was lower than both C and DX groups, which would support the notion that decreased IHR masked the effects of sympathetic overactivity in the current study. Further, it supports evidence that STZ-induced diabetes may have a direct effect on heart rate by modifying the heart itself [
An important consideration regarding the design of the current study was the use of anesthetized rats. In order to accurately reflect cardiovascular parameters in such a state, we selected an anaesthetic regime that provides the lowest level of influence on baseline and reflexive CV control attainable in rodent models [
In this study, T1DM induced indications of parasympathetic withdrawal, sympathetic overactivity, and, despite a decreased IHR, no change in resting MAP or HR. However, concurrent exercise training with T1DM maintained the sensitivity of the parasympathetically mediated baroreflex bradycardia, prevented an increase in vascular sympathetic tone, maintained a higher bodyweight, and prevented a decrease in IHR. The ability of exercise training to preserve parasympathetic function in this model of T1DM indicates that the exercise-mediated improvements to parasympathetic function are independent of alterations in [BG]. However, the finding that [NPY] remained elevated suggests that hyperglycemia has a direct impact on adrenergic activity. Taken together, our T1DM model of progressive STZ induction and insulin treatment induced autonomic impairments similar to those observed in clinical T1DM and demonstrates the novelty of this model for investigating the effectiveness of high intensity aerobic exercise training as a means to prevent the progression of CAN in T1DM. Thus, although not examined in this study, the mechanisms that underlie the physiological changes caused by T1DM and exercise can be the focus of future investigations using this model.
Blood glucose
Blood pressure
Baroreflex sensitivity
Sedentary control
Cardiovascular autonomic neuropathy
Exercised control
Cardiovascular
Sedentary T1DM
Exercised T1DM
High frequency
Heart rate
Heart rate variability
Intrinsic heart rate
Mean arterial pressure
Neuropeptide Y
Phenylephrine
Systolic blood pressure
Sodium nitroprusside
Sympathetic nervous system
Streptozotocin
Type 1 diabetes mellitus.
The authors declare that there is no conflict of interests regarding the publication of this paper.
Kenneth N. Grisé contributed to the study design, data collection, data analysis, and writing. T. Dylan Olver, Matthew W. McDonald, Adwitia Dey, and Mao Jiang contributed to the study design and data collection. James C. Lacefield and Earl G. Noble facilitated the data collection. J. Kevin Shoemaker provided data analysis and writing. C. W. James Melling contributed to the study design, data analysis, and writing.
This study was supported by the Canadian Institute of Health Research Grant (nos. CCT-83029 and 217532) and the Natural Sciences and Engineering Council Discovery Grant (RGPGP-2015-00059).