Sorbitol accumulation is postulated to play a role in skeletal muscle dysfunction associated with diabetes. The purpose of this study was to determine the effects of insulin and of endurance exercise on skeletal muscle sorbitol levels in streptozotocin-induced diabetic rats. Rats were assigned to one of five experimental groups (control sedentary, control exercise, diabetic sedentary, diabetic exercise, diabetic sedentary no-insulin). Diabetic rats received daily subcutaneous insulin. The exercise-trained rats ran on a treadmill (1 hour, 5X/wk, for 12 weeks). Skeletal muscle sorbitol levels were the highest in the diabetic sedentary no-insulin group. Diabetic sedentary rats receiving insulin had similar sorbitol levels to control sedentary rats. Endurance exercise did not significantly affect sorbitol levels. These results indicate that insulin treatment lowers sorbitol in skeletal muscle; therefore sorbitol accumulation is probably not related to muscle dysfunction in insulin-treated diabetic individuals. Endurance exercise did not influence intramuscular sorbitol values as strongly as insulin.
Diabetes mellitus (DM) is an endocrine condition characterized by hyperglycemia and is associated with vascular and neurological complications. Common components of diabetes management include insulin and physical exercise. A significant reduction in muscle strength is observed in patients with long-term type 1 DM with severe distal motor neuropathy playing a major role [
In experimental diabetes, significant alterations in contractile properties of skeletal muscle have been described in [
Insulin decreases intracellular sorbitol by deviating glucose away from the polyol pathway and metabolizing it through nonpolyol metabolic pathways. Modifications in the metabolism of glucose induced by endurance exercise training have the potential to affect the concentrations of sorbitol in diabetic individuals too. For example, endurance exercise training increases the synthesis and activity of hexokinase II [
The main purpose of this study was to determine the effect of insulin treatment in STZ-induced diabetes on skeletal muscle sorbitol values. Our goal was to mimic the conditions observed in clinical settings by administering insulin to the STZ-induced diabetic rats. We hypothesized that insulin administration would significantly decrease sorbitol accumulation in skeletal muscle. A secondary purpose was to determine the benefits of endurance exercise training on skeletal muscle sorbitol accumulation in STZ-induced diabetes. We hypothesized that exercise would also significantly decrease sorbitol accumulation in skeletal muscle.
The University of Minnesota Institutional Animal Care and Use Committee approved the experimental procedures, and the Principles of Laboratory Animal Care according to NIH publications were followed. Thirty-nine male Sprague-Dawley rats (Charles River, Wilmington, MA) were used in this study. They were housed in a light (12 hour light : dark cycle) and temperature (
Diabetes was induced with intravenous administration of streptozotocin (Sigma, St. Louis, MO), 60 mg/kg of body weight, in a saline solution. Control rats were injected only with saline solution. Diabetes was confirmed if serum glucose concentration was at least 250 mg/dl on two consecutive days, with glucose testing beginning on the second day post-STZ administration. Thereafter, in the diabetic animals, serum glucose was measured three times per week for the following 12 weeks using the glucose oxidase method (Glucose Analyzer II, Beckman Instruments, Palo Alto, CA). Subcutaneous NPH insulin (Neutral Protamine Hagedorn) was administered daily to diabetic rats at the beginning of their active phase in the evening, according to a sliding scale based on serum glucose levels. Insulin was given to mimic the clinical condition of patients, and to prevent severe metabolic complications, but not to provide tight glucose control. In the diabetic rats not receiving insulin, mean glucose value was
Mean blood glucose, dose of NPH daily insulin administration, and final body weight by group. Data are expressed as means
Groups ( | Blood glucose (mg/dl) | NPH Insulin (IU) | Body weight (g) | EDL (g) | SOL (g) |
---|---|---|---|---|---|
Diabetes sedentary no insulin, DSNI (6) | — | ||||
Diabetic sedentary, DS (9) | 0.226 + 0.03 | ||||
Diabetic exercise, DE (8) | |||||
Control sedentary, CS (8) | — | ||||
Control exercise, CE (8) | — |
Rats assigned to the exercise groups ran on a treadmill five times per week for 60 minutes, starting at a speed of 15 m/min, and progressively increasing speed by two to three m/min every two weeks up to a speed of 27 m/min [
To assess training status, blood lactates were determined at the end of the 12-week training period. Blood lactate was measured using a YSI lactate analyzer (YSI 2300 STAT PLUS. Yellow Springs, OH). Blood samples (20
To assess cellular aerobic capacity in muscles with different skeletal muscle myosin heavy chain isoforms (fiber type), citrate synthase activity was determined in the white (superficial region) and red (deep lateral region) gastrocnemius muscle from the same animals using the method described by Srere P. A. [
The exercise-trained animals were killed four to six days after their last bout of running. This timing was chosen to avoid acute effects of the last bout of exercise. At the end of 12 weeks, all groups of rats were deeply anesthetized with pentobarbital sodium (50 mg/kg of body weight i.p.). The extensor digitorun longus (EDL) and the soleus (SOL) muscles were excised, weighed, frozen in liquid nitrogen, and stored in
Each muscle was cut into small pieces and ground in the presence of liquid nitrogen until it had a powdered appearance. The weight of the powered muscle was determined, after which 1 mL of ice-cold 0.5 M perchloric acid was added and the samples were sonicated to disrupt the tissue. The sonicated muscle was centrifuged for five minutes to remove precipitate. The resulting supernatant was neutralized by adding a solution containing one part of tri-n-octylamine and three parts of 1,1,2-trichlorotrifluoroethane. The solution was then vortexed for 45 seconds, then centrifuged for five minutes at 7000 rpm. The upper layer was recovered and adjusted to pH 7.0 with 2 M Tris base, and the samples were evaporated to dryness in a Speed-Vac concentrater and stored in
The dried neutralized muscle extract was redissolved in 75
Descriptive statistical analysis included mean and SEM. A one way ANOVA was used to determine differences in blood lactate values and if a significant overall
Sorbitol data from the EDL and SOL muscles were normalized by log transformation and that data were used for statistical analysis. However, for comparison with the existing literature, sorbitol values in
Table
Muscle weights for the EDL and SOL were 72% lower in the DSNI group than in the other groups (
Following 12 weeks of endurance exercise training, the mean difference in blood lactate values between the resting state and the postacute exercise state was significantly lower in the exercised rats than in the sedentary rats (Figure
Exercise training induced adaptations. (a) Change in blood lactate values after 10 minutes of treadmill running. Values are in mmol/L
Sorbitol levels were higher in diabetic rats not receiving insulin than in the other experimental groups (Figure
Effects of insulin administration on muscle sorbitol levels. Data are expressed as
In this study, endurance training in the diabetic rats did not reduce sorbitol levels to the degree that significance was obtained at the predetermined
Effect of endurance training on muscle sorbitol levels. Data are expressed as
This study shows the effects of insulin and endurance exercise training on sorbitol content in skeletal muscle of diabetic rats. We provide evidence that the administration of insulin to diabetic rats is sufficient to lower sorbitol content to values observed in control rats. Additionally, in insulin-treated diabetic rats, endurance exercise training showed a tendency to add to the sorbitol lowering effects of insulin although not to the preselected level of statistical significance (
As expected, STZ-induced diabetes increased sorbitol concentrations in both EDL and SOL muscles. This elevation was significantly lowered by insulin. Administration of insulin at a dosage that was not aimed at restoring blood glucose to normal levels, but at preventing severe metabolic complications, was sufficient to lower muscle sorbitol values in diabetic rats to values observed in nondiabetic controls. Insulin most likely exerts its sorbitol-lowering effect by increasing the activity of hexokinase II, an insulin-dependent enzyme [
With diabetes there is muscle impairment, but the underlying mechanisms remain unknown. The hypothesized potential mechanisms to explain this impairment include: diabetic motor neuropathy [
Endurance exercise training appears to lower sorbitol values in the two muscles investigated in this study (EDL and SOL). The mechanism responsible for lowering sorbitol is probably related to the acute and long-term effects of exercise on glucose metabolism. The increase in citrate synthase activity and the lower blood lactate values in the endurance-trained rats are evidence that metabolic adaptations occurred secondary to the endurance training protocol. Such adaptations have previously been described to include an increase in hexokinase II activity that may divert glucose from the polyol pathway [
The findings in the current study suggest that sorbitol accumulation does not appear to be responsible for the functional alterations observed in muscles of diabetic rats. An alternative explanation could be that the polyol pathway induces alterations in the redox state of the muscle, increasing deleterious oxidative stress [
Rats in this study were killed four–six days after their last bout of exercise. This timing might have influenced the results because the acute effects of exercise on glucose uptake and metabolism lasts at most for 40 hours [
In summary, we have determined that insulin treatment of diabetic rats significantly reduces skeletal muscle sorbitol concentration. Moderate-to-high intensity endurance exercise training may further decrease sorbitol accumulation, but results are less conclusive. Therefore, the results of this study seem to indicate that sorbitol accumulation is most likely not related to skeletal muscle dysfunction in insulin-treated diabetes.
The authors thank Lilly Yee, Paul Scott, and Chris Ostrom for their technical assistance in the determination of sorbitol. They also thank Christopher P. Elder for his helpful comments in the discussion of this manuscript, and the Diabetes Institute at the University of Minnesota. This study was partially funded by grants from the Center on Aging and from the Center on Muscle and Muscle Disorders at the University of Minnesota. The doctoral scholarship was provided by the Universidad de Oriente, Venezuela.