LeAnn M. Snow

Advanced Glycation End Product in Diabetic Rat Skeletal Muscle in vivo

Background: Advanced glycation end products (AGEs) are implicated in the etiology of diabetic complications in the kidney, nerve and eye. Skeletal muscle contractile parameters have also been found to be altered in diabetes. Glycation has not been extensively studied in skeletal muscle, but AGE-modified proteins may influence contractility. Objective and Methods: The aim of this study was to use immunohistochemistry to identify distribution patterns of the AGE NΕ-(carboxymethyl)-lysine in plantaris muscle of diabetic rats. Results: Results revealed the presence of NΕ-(carboxymethyl)-lysine intracellularly and also at sites along the myofiber periphery. The number of myofibers immunolabeling for AGE in animals with diabetes was more than 4-fold greater than in control animals. Additionally, there was a greater proportion of slow + fast myosin heavy chain coexpression in the AGE-positive cells from diabetic animals than in AGE-positive fibers from control animals. No significant difference was present between cross-sectional areas of AGE-positive fibers and AGE-negative fibers within the respective experimental groups. Conclusions: AGE accumulation is greater in skeletal muscle in vivo from diabetic animals than in control animals. This AGE accumulation appears to be associated with fiber-type transformation rather than with myofiber size. Further study is needed to determine the identity of these AGE-modified proteins and to determine how they influence skeletal muscle function in diabetes.

Effect of endurance exercise on myosin heavy chain isoform expression in diabetic rats with peripheral neuropathy.

Snow LM, Sanchez OA, McLoon LK, Serfass RC, Thompson LV: Effect of endurance exercise on myosin heavy chain isoform expression in diabetic rats with peripheral neuropathy. Am J Phys Med Rehabil 2005;84:770–779. Objective:This study evaluated the effect of endurance exercise on myosin heavy chain (MHC) isoform expression in soleus muscle of diabetic rats with peripheral neuropathy Design:Male Sprague Dawley rats were randomly divided into four groups: control sedentary, diabetic sedentary, control exercise, and diabetic exercise. The exercised animals performed treadmill running five times per week. After 12 wks, electrophysiologic testing documented peripheral neuropathy in the diabetic rats. The soleus muscles were then excised and quick-frozen. Cross-sections were immunohistochemically stained for slow, fast, developmental, and neonatal MHCs. Fiber-type composition and fiber cross-sectional areas were then determined. Results:The diabetic groups showed a significantly greater percentage of fast MHC than did the control groups, regardless of exercise status (diabetic sedentary, 22.6%; diabetic exercise, 25.2%; control sedentary, 13.5%; control exercise, 13.1%). The diabetics also showed a significantly lower percentage of slow-only MHC than controls (diabetic sedentary, 77.1%; diabetic exercise, 74.3%; control sedentary, 86.2%; control exercise, 86.1%). No differences in muscle fiber cross-sectional area existed between the groups. The exercised animals showed greater expression of developmental MHC than did the sedentary animals (diabetic sedentary, 1.6%; diabetic exercise, 3.8%; control sedentary, 0.8%; control exercise, 2.0%). Conclusion:The altered slow and fast MHC expression in the diabetic muscle is similar to MHC expression in several other conditions, including decreased neuromuscular activity and denervation. Mechanisms of this MHC expression shift are unknown. Chronic endurance training does not alter adult MHC expression in the diabetic animals. The developmental MHC expression is likely a manifestation of uphill treadmill running due to eccentric contractions in the soleus resulting in myofiber injury and regeneration.

Influence of Insulin and Muscle Fiber Type in Nε-(Carboxymethyl)-Lysine Accumulation in Soleus Muscle of Rats with Streptozotocin-Induced Diabetes Mellitus

Background: Nε-(carboxymethyl)-lysine (CML) is an advanced glycation end product (AGE), the accumulation of which has been implicated in the etiology of diabetes complications. Skeletal muscle in diabetes demonstrates altered function, and increased accumulation of CML has been found in several fast-twitch muscles of diabetic animals. Objective: This study aims to explore the accumulation of CML in soleus (a slow muscle) in diabetic animals, with and without insulin therapy. Methods: Twenty-one rats were randomly divided into control and diabetes groups (DNI: diabetes without insulin; DI: diabetes with insulin; C: control). Diabetes was induced by intravenous administration of streptozotocin. At the end of the 12-week experimental period the soleus muscle was excised and snap frozen in liquid nitrogen. Muscle cross-sections were immunolabeled for CML. The number of CML-labeled muscle fibers was quantified; fibers were also evaluated for fiber types and cross-sectional areas. Results: The percentage of myofibers immunolabeling for CML was highest in the DNI group (13.8 ± 2.5%), lower in the DI group (5.4 ± 1.1%) and lowest in the C group (2.1 ± 0.6%). Statistical analysis revealed that AGE accumulation was significantly greater in the DNI group than in both C and DI groups (p = 0.0002). There was no significant difference between C and DI groups. In the DNI animals, AGE-positive myofibers showed a higher percentage of fast fiber types than did the AGE-negative fibers (49.5 ± 6.9 vs. 13.7 ± 1.5%, p = 0.002). No differences existed in cross-sectional areas between AGE-positive and AGE-negative fibers within any group. Conclusion: The greatest accumulation of AGE was in the soleus of the DNI group, and was significantly less in the DI group. These findings may be linked to disordered glucose metabolism, increased oxidative stress and/or fiber type transformation in these muscles.

Skeletal muscle plasticity after hemorrhagic stroke in rats: influence of spontaneous physical activity.

ObjectiveThe aim of this study was to determine the contribution of spontaneous post-stroke physical activity to skeletal muscle plasticity after stroke. DesignA randomized controlled study was conducted of 24 young adult male Sprague-Dawley rats assigned to three experimental groups: (1) STR—hemorrhagic stroke in the right caudoputamen; (2) SHAM—procedural control; and (3) CONT—no intervention (n = 8/group). Neurologic testing was performed before and 2 wks after stroke. Spontaneous physical activity was monitored five nights per week for 1 wk preoperatively and 2 wks postoperatively. Two weeks after stroke induction, bilateral soleus and tibialis anterior muscles were harvested. Myofiber cross-sectional areas were determined, and fiber typing was performed with immunohistochemistry. ResultsSTR animals demonstrated neurologic deficit in the contralesional hindlimb 2 wks after stroke. Quantity of spontaneous physical activity did not differ between groups within each of the week-long study intervals. No significant difference was found in fiber types or cross-sectional areas in the soleus muscle of STR vs. CONT groups. However, the tibialis anterior muscle of the contralesional hindlimbs of the STR animals showed atrophy in 2x and 1 + 2x myofibers, as well as type 1 hypertrophy. ConclusionsSkeletal muscle adaptation occurs by 2 wks post-stroke in this model. It is muscle specific and appears to be influenced by factors other than spontaneous post-stroke physical activity.

Skeletal Muscle Sorbitol Levels in Diabetic Rats with and without Insulin Therapy and Endurance Exercise Training

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 experimental group (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.

Poster 46: Immunohistochemical evidence of immature myosin expression in aged rat skeletal muscle1

Abstract Objective: To evaluate 2 hindlimb skeletal muscles in young and aged rats for presence of immature myosin heavy chain (MHC) isoforms. Design: Randomized controlled trial. Setting: Basic science laboratory. Animals: Fisher 344 Brown Norway F1 hybrid rats, ages 12 and 36 months. Interventions: The soleus and extensor digitorum longus (EDL) muscles of young (12mo old) and aged (36mo old) sedentary rats were excised, quick frozen, and stained immunohistochemically for the developmental MHC isoform. Muscle cross-sections were then evaluated for the number of developmental positive fibers per unit area. Main Outcome Measures: Number of muscle fibers per unit cross-sectional area expressing developmental MHC. Results: The soleus muscles of the aged rats showed significantly greater numbers of developmental MHC positive cells when compared with the soleus of the 12-month-old rats. The EDL of the 36-month-old rats exhibited noticeably greater numbers of developmental MHC positive cells than in the young animals in half of the muscles studied. Conclusions: There is evidence of skeletal muscle regeneration occurring in the soleus of the aged rats in the midst of the fiber atrophy that accompanies aging. Such changes in the EDL also occur, but appear to be less common. The muscle specific differences observed in the expression of developmental MHC with age may be influenced by the fiber type and function differences of this muscle as compared with the soleus. Evaluation of the coexpression of the developmental MHC with the mature MHCs (slow, fast) is currently under investigation.