Histochemical properties of skeletal muscle fibers in streptozotocin-diabetic rats

Summary The response of rat gastrocnemius muscle fibers to chronic streptozotocin-diabetes was studied. Transverse sections of this muscle from normal and diabetic rats were histochemically assayed for reduced diphosphopyridine nucleotide-diaphorase, myofibrillar adenosine triphosphatase, mitochondrial alpha-glycerophosphate dehydrogenase, beta-hydroxybutyrate dehydrogenase, and alkaline phosphatase activities. Cross-sectional areas of the fiber types were measured, and fiber capillarization and populations estimated. Chemically-induced diabetes appeared to have little effect on the metabolic or morphological properties of slow-twitch fibers. However, a general dedifferentiation occurred in the 2 fast-twitch fiber populations. There was a loss of oxidative potential in the fast-twitch-oxidative-glycolytic fibers, and a significant decrease in size in the fast-twitch-glycolytic fibers. No change in the proportions of slow- and fast-twitch fibers in the muscles of diabetic rats occurred. It is concluded that hypoinsulinism has differential effects on the 3 fiber types in heterogeneous rat skeletal muscle, and that slow-twitch fibers are least affected by the diabetic condition.     

Metabolic alterations in skeletal muscle of chronically streptozotocin-diabetic rats

This study was accomplished to determine the effects of chronic streptozotocin diabetes and insulin treatment on selected enzymes and substrates used in energy transduction in muscles composed of different muscle fiber types. Triglyceride concentration in all the muscles of diabetic rats was significantly elevated. Glycogen and protein concentrations were unchanged. The enzyme activities of hexokinase and alanine aminotransferase were significantly reduced and 3-hydroxyacyl-CoA dehydrogenase increased in all the muscles. Declines in phosphofructokinase, lactate dehydrogenase, citrate synthase, and succinate dehydrogenase activities were found in the red gastrocnemius and plantaris. Glycerol-3-phosphate dehydrogenase activity was lower than normal in the red gastrocnemius. Insulin treatment to the diabetic rats returned the altered triglyceride content and enzyme activities to normal, with exception of the lower alanine aminotransferase activity in the red gastrocnemius and plantaris. However, this enzyme was significantly ameliorated when compared with the untreated diabetic rats. The findings show that hypoinsulinism has a differential effect on the enzymatic profile of the different skeletal muscle fiber types, with those of the red gastrocnemius being most severely affected. Insulin treatment returned the enzymatic profile of the fiber types in diabetic rats to essentially normal.     

Type I diabetes affects skeletal muscle glutamine uptake in a fiber-specific manner

Skeletal muscle serves as the body's major glutamine repository, and releases glutamine at enhanced rates during diabetes, but whether all muscles are equally affected is unknown. System N(m) activity mediates most trans-sarcolemmal glutamine movement, and although two System N (SN) isoforms have been identified (SN1/sodium-coupled neutral amino acid transporter or System N and A transporters [SNAT]-3; and SN2/SNAT5), their expression in skeletal muscle remains controversial. Here, the impact of Type I diabetes on glutamine uptake and System N transporter expression were examined in fast- and slow-twitch skeletal muscle from spontaneously diabetic (BB/Wor-DP) rats. Net glutamine uptake in fast-twitch fibers was decreased 75%-95%, but enhanced more than 2-fold in slow-twitch muscle from diabetic animals relative to nondiabetic controls. Both SNAT3 and SNAT5 mRNA were expressed in both muscle fiber types and their abundance was unaffected by diabetes. This represents the first report of differential fiber-specific effects of diabetes on skeletal muscle glutamine transport and the co-expression of distinct System N transporters in skeletal muscle.     

Decay of succinate dehydrogenase activity in rat skeletal muscle following streptozotocin injection.

Succinate dehydrogenase activities of 4 skeletal muscles with widely divergent fiber compositions were determined in rats that recieved either 70 or 80 mg streptozotocin per kg body wt at various time intervals following injection. Enzyme activities in all muscles declined to a lower final level and exhibited a more rapid decay in animals receiving the larger dosage. White gastrocnemius muscle, which contains no slow-twitch fibers, experienced the greatest relative loss of activity, and soleus muscle, possessing the highest proportion of slow-twitch fibers, the smallest loss. The findings indicate that extrapancreatic effects of the drug may have been partially responsible for the differential effects of the 2 dosages on the muscles.     

Loss of fast-twitch isomyosins in skeletal muscles of the diabetic rat.

By means of pyrophosphate electrophoresis the myosin isoenzyme pattern of two fast-twitch skeletal muscles (extensor digitorum longus, gastrocnemius) and one slow-twitch muscle (soleus) was investigated in control rats and was compared with that of rats 4 weeks after induction of diabetes mellitus by streptozotocin injection. In the fast-twitch muscles the isomyosin pattern consisting of FM1 (fast isomyosin 1), FM2 and FM3 was strongly affected by diabetes, resulting in an extensive loss of FM1 and a substantial decrease of FM2. These changes were also apparent when the light chains of the fast isomyosins were analysed by two-dimensional electrophoresis: LC3f (myosin light chain 3f) largely disappeared and LC2f was significantly diminished. In contrast, the isomyosin pattern in soleus muscle, consisting of SM1 (slow isomyosin 1) and SM2, was not affected by the diabetic state, and two-dimensional electrophoresis revealed a normal light-chain pattern of LC1sa, LC1sb and LC2s. These results indicate that the isomyosins of slow-twitch oxidative myofibres are more resistant to the hormonal and metabolic disorders during diabetes mellitus than are the isomyosins of fast-twitch fibres.     

Ultrastructural alterations in skeletal muscle fibers of streptozotocin-diabetic rats

Summary The ultrastructure of fast-twitch-oxidative-glycolytic (FOG), fasttwitch-glycolytic (FG) and slow-twitch-oxidative (SO) fibers in plantaris and soleus muscles of normal and streptozotocin-diabetic rats was studied. In the diabetic animals, the mitochondria of FOG and SO fibers showed a loss of cristae and an increase in electron-dense granules. There was also an increased number of lipid droplets in close proximity to the mitochondria and the nuclei, and a separation of individual muscle nuclei to form satellite cells. Higher incidences of surface projections and sarcoplasmic splittings at the nuclear region were noticed in SO fibers. The FG fibers showed some disorientation of the T-tubular system. It is concluded that streptozotocin-diabetes has differential effects on the fine structure of the three fiber types of rat skeletal muscle.Supported by USPHS Grant AM 18280-04, Boston University Grant GRS-405-BI, and a grant-in-aid award from Sigma Xi Society     

Alanine and aspartate aminotransferase activities in muscles of diabetic rats

Activities of alanine and aspartate aminotransferase in different muscle types and in the liver of streptozotocin diabetic rats were studied 1,2 and 3 days after administering of streptozotocin. It was shown that the activity of both enzymes was elevated in the "white" layer of the vastus lateralis, in the liver and in the heart, whereas it remained unchanged in the "red" layer of the same muscle, in the soleus and the diaphragm. It is concluded that the effect of acute insulin deficiency on the aminotransferase activity in skeletal muscles depends on the muscle fiber composition and does not appear in muscles with a high oxidative potential. These results indicate that muscle fiber composition should be taken into account when evaluating the role of insulin in amino acid metabolism in the muscle.     

Multiple stimulations for muscle–nerve–blood vessel unit in compensatory hypertrophied skeletal muscle of rat surgical ablation model

Tissue inflammation and multiple cellular responses in the compensatory enlarged plantaris (OP Plt) muscle induced by surgical ablation of synergistic muscles (soleus and gastrocnemius) were followed over 10 weeks after surgery. Contralateral surgery was performed in adult Wistar male rats. Cellular responses in muscle fibers, blood vessels and nerve fibers were analyzed by immunohistochemistry and electron microscopy. Severe muscle fiber damage and disappearance of capillaries associated with apparent tissue edema were observed in the peripheral portion of OP Plt muscles during the first week, whereas central portions were relatively preserved. Marked cell activation/proliferation was also mainly observed in peripheral portions. Similarly, activated myogenic cells were seen not only inside but also outside of muscle fibers. The former were likely satellite cells and the latter may be interstitial myogenic cells. One week after surgery, small muscle fibers, small arteries and capillaries and several branched-muscle fibers were evident in the periphery, thus indicating new muscle fiber and blood vessel formation. Proliferating cells were also detected in the nerve bundles in the Schwann cell position. These results indicate that the compensatory stimulated/enlarged muscle is a suitable model for analyzing multiple physiological cellular responses in muscle–nerve–blood vessel units under continuous stretch stimulation. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Effect of diabetes on guanine nucleotide exchange factor activity in skeletal muscle and heart

The present study examined the effects of diabetes and insulin treatment of diabetic rats on the activity of the protein synthesis initiation factor, the guanine nucleotide exchange factor. In extracts from gastrocnemius and psoas muscles from two-day diabetic rats, guanine nucleotide exchange factor activity was reduced to 80% and 67% of control values, respectively. Insulin treatment (2 h) restored guanine nucleotide exchange factor activity to control values in both muscles. In contrast, guanine nucleotide exchange factor activity was unchanged in extracts from either soleus muscle or heart from diabetic rats compared to controls. Also, insulin treatment did not increase guanine nucleotide exchange factor activity in extracts from soleus and heart. The results suggest that the diabetes-induced impairment in peptide-chain initiation in fast-twitch skeletal muscle (i.e. gastrocnemius and psoas) is related to an inhibition of guanine nucleotide exchange factor activity and that slow-twitch muscle is spared from the effect on initiation due to the preservation of guanine nucleotide exchange factor activity.     

The role of insulin in the control of triacyglycerol (TG) in the rat skeletal muscles

The role of insulin in the control of triacyglycerol (TG) in different types of skeletal muscle has not been fully recognized so the aim of the present study was to fill this gap. The experiments were carried out on control rats, those fed with olive oil or fed with the oil and treated with insulin and on streptozotocin diabetic animals at rest and there after exercise till exhaustion. The level of TG was measured in the white and red layers of the vastus lateralis, the soleus and the diaphragm. It was found that acute feeding with olive oil had no effect on TG level in either muscle type examined. Insulin administered to rats fed with oil increased TG level in the red vastus. Streptozotocin diabetes caused an increase in TG level in muscles with high oxidative potential. Exercise lowered the level of TG only in the red vastus of the diabetic rats. It is concluded that insulin may increase muscle TG level. Accumulation of TG in muscles of rats with acute diabetes is likely to be a result of the high plasma free fatty acid concentration. Acute insulin deficiency did not affect the muscle TG response to exercise.     

Ultrastructural localization of lectin receptors on the bone-marrow sinusoidal endothelium of the rat

The purpose of this study was to estimate the absolute and relative masses of the three types of skeletal muscle fibers in the total hindlimb of the male Sprague-Dawley rat (Rattus norvegicus). For six rats, total body mass was recorded and the following weights taken from dissection of one hindlimb: 32 individual major muscles or muscle parts, remaining skeletal musculature (small hip muscles and intrinsic foot muscles), bone, inguinal fat pad, and skin. The fibers from the 32 muscles or muscle parts (which constituted 98% of the hindlimb skeletal muscle mass) were classified from histochemistry as fast-twitch oxidative glycolytic (FOG), fast-twitch glycolytic (FG), or slow-twitch oxidative (SO), and their populations were determined. Fiber cross-sectional areas from the same muscles were measured with a digitizer. Mass of each of the fiber types within muscles and in the total hindlimb was then calculated from fiber-type population, fiber-type area, and muscle-mass data. Skeletal muscle made up 71% of the total hindlimb mass. Of this, 76% was occupied by FG fibers, 19% by FOG fibers, and 5% by SO fibers. Thus, the FG fiber type is clearly the predominant fiber type in the rat hindlimb in terms of muscle mass. Fiber-type mass data are compared with physiological (blood flow) and biochemical (succinate dehydrogenase activities) data for the muscles taken from previous studies, and it is demonstrated that these functional properties are closely related to the proportions of muscle mass composed of the various fiber types.     

Proximal skeletal muscle alterations in streptozotocin-diabetic rats: a histochemical and morphometric analysis.

The response of rat quadriceps muscle fibers to chronic streptozotocin (STZ) diabetes was studied. Transverse sections of rectus femoris muscle from diabetic and weight-matched control rats were assayed for myofibrilar adenosine triphosphatase (ATPase) and nicotinamide adenine dinucleotide-tetrazolium reductase (NADH-TR). A quantitative analysis was carried out by an automatic interactive analysis system focused on the fiber type size and distribution. STZ-induced diabetes caused important effects in this muscle, with changes in the distribution of oxidative enzyme reactions, type I fiber hypertrophy, and type II fiber atrophy, which was greater in type IIB than in type IIA. It is concluded that hypoinsulinism produces morphological alterations in proximal skeletal muscle fibers that are similar to those of neurogenic myopathy. Thus the pathological changes in these mammalian muscle fibers could explain the clinical syndrome seen in diabetic patients called "diabetic symmetrical proximal motor neuropathy," perhaps the least understood of the major neuropathic complications of diabetes.     

Rat muscle protein turnover and redox state in progressive diabetes

Protein synthesis and degradation, and redox state were measured in soleus and extensor digitorum longus muscles of rats up to 12 days after injection of streptozotocin. Muscle growth was slower in these animals apparently due to slower protein synthesis throughout the duration of diabetes. Up to day 4 after injection of streptozotocin or withdrawal of insulin from treated, diabetic animals, the muscle ratio of lactate/pyruvate, an indicator of the cytoplasmic NAD+ redox couple, was lower and protein degradation was faster than in control muscles. Thereafter, the ratio of lactate/pyruvate was greater and protein degradation was slower than in size- or age-matched control muscles. Insulin treatment in vitro or in vivo increased lactate/pyruvate and decreased proteolysis. Therefore, in muscles of streptozotocin-diabetic rats, the initial increase and later fall in proteolysis, and the inhibition of proteolysis by insulin, may correlate with opposite changes in NADH/NAD+.     

Effect of acute streptozotocin diabetes on fatty acid content and composition in different lipid fractions of rat skeletal muscle.

The aim of the present study was to examine the effect of acute streptozotocin diabetes on long chain fatty acid content and composition in different lipid classes of particular muscle types in the rat. Two days after streptozotocin administration, rats were anesthetised, and the white and red sections of the gastrocnemius, the soleus and the blood were taken. Lipids were extracted with chloroform/methanol and separated into different fractions (phospholipids, free fatty acids, di- and triacylglycerols) by means of thin layer chromatography. Fatty acids of each fraction were identified and quantified by means of gas-liquid chromatography. The diabetes resulted in elevation of the concentration of blood glucose (over four-fold) and the plasma free fatty acid (over two-fold). Total free fatty acid content in the muscles of diabetic rats increased by 26% in the white, 24% in the red gastrocnemius and 21% in the soleus. There were also changes in the composition of that fraction in each muscle. Diacylglycerol fatty acid content was elevated in both parts of the gastrocnemius (the white part by 15%, the red part by 44%) and remained stable in the soleus of the diabetic rats. The content of triacylglycerol fatty acids was elevated only in the red gastrocnemius in the diabetic group (by 112%), but changes in fatty acid composition in this fraction occurred in each muscle. The content of phospholipid fatty acids was elevated in the white gastrocnemius (by 13%) and remained stable in other muscles. There were only minor changes in phospholipid fatty acid composition in the diabetic rats. We concluded that acute insulin deficiency changes fatty acid content and composition in skeletal muscle lipids. The changes depend both on lipid fraction and muscle type.     

Fiber type changes in rat skeletal muscle after intense interval training

Female Sprague-Dawley rats were subjected to a ten week training program to determine the influence of intense interval running on the fiber type composition of selected hindlimb muscles; soleus (S), plantaris (P), deep vastus lateralis (DVL), and superficial vastus lateralis (SVL). The muscles of one hindlimb were used for histochemical ATPase analysis to determine the distribution of fiber types and those of the contralateral hindlimb were assayed biochemically for citrate synthase activity (an aerobic marker). Training induced a significant increase in citrate synthase activity in each muscle section. The largest absolute increase occurred in the DVL and the largest relative increase occurred in the SVL. The distribution of fiber types within the S (85% slow-twitch) and SVL (100% fast-twitch) remained unchanged with training. However, significant increases in the percentage of type I (slow-twitch) fibers in both the P (2-fold) and DVL (3-fold) were observed with concomitant decreases in the type II (fast-twitch) population. In addition, training induced significant changes in the fast-twitch subtype populations of the DVL (IIB----IIA). These data suggest exercise-induced fiber type transformations occurring both within the fast-twitch population and between fast-twitch and slow-twitch fibers in certain hindlimb muscles of the rat following a high intensity interval training program.     

Lactate dehydrogenase isoenzyme spectrum of fast and slow muscles with innervation disorders

A relative content of muscle fibers of various types and the spectrum of lactate dehydrogenase (LDH) isozymes were studied in fast-twitch (extensor digitorum longus) and slow-twitch (soleus) muscles of newborn rats, of those aged 2, 3 weeks and one month and of adult rats after neonatal sciatic denervation and application of 0.5 mM colchicine solution to the sciatic nerve. No muscle fibers of various types were found (from the level of succinate dehydrogenase activity) in one-month-old rats, whereas the control and fast-twitch muscles showed A, B and C types and the slow-twitch one B and C types. The denervation brought about an increase in the content of LDH4 and LDH5 in both the muscles, while colchicine application gave rise to an increase in LDH2 activity, diminution of LDH1 in the fast-twitch muscle and elevation of LDH4 in the slow-twitch one. The data obtained attest to the retardation of muscle differentiation under application of the colchicine-induced blockade of axoplasmic transport.     

Effects of endurance exercise-training on single-fiber contractile properties of insulin-treated streptozotocin-induced diabetic rats.

The purpose of this study was to characterize the contractile properties of individual skinned muscle fibers from insulin-treated streptozotocin-induced diabetic rats after an endurance exercise training program. We hypothesized that single-fiber contractile function would decrease in the diabetic sedentary rats and that endurance exercise would preserve the function. In the study, 28 rats were assigned to either a nondiabetic sedentary, a nondiabetic exercise, a diabetic sedentary, or a diabetic exercise group. Rats in the diabetic groups received subcutaneous intermediate-lasting insulin daily. The exercise-trained rats ran on a treadmill at a moderate intensity for 60 min, five times per week. After 12 wk, the extensor digitorum longus and soleus muscles were dissected. Single-fiber diameter, Ca(2+)-activated peak force, specific tension, activation threshold, and pCa(50) as well as the myosin heavy chain isoform expression (MHC) were determined. We found that in MHC type II fibers from extensor digitorum longus muscle, diameters were significantly smaller from diabetic sedentary rats compared with nondiabetic sedentary rats (P < 0.001). Among the nondiabetic rats, fiber diameters were smaller with exercise (P = 0.038). The absolute force-generating capacity of single fibers was lower in muscles from diabetic rats. There was greater specific tension (force normalized to cross-sectional area) by fibers from the rats that followed an endurance exercise program compared with sedentary. From the results, we conclude that alterations in the properties of contractile proteins are not implicated in the decrease in strength associated with diabetes and that endurance-exercise training does not prevent or increase muscle weakness in diabetic rats.     

Compensatory adaptations of skeletal muscle fiber types to a long-term functional overload

We investigated selected histochemical and histometrical characteristics of the heterogeneous fiber types of rat skeletal muscle following long-term compensatory muscle growth. Sixty days following surgical removal of the synergistic gastrocnemius muscle, the compensated ipsilateral plantaris and soleus muscles and the corresponding control muscles from the contralateral leg were excised and stained histochemically for myofibrillar ATPase and DPNH-diaphorase activities. The number of fibers per cross-section was determined by a direct count from transverse sections taken from the midportion of the muscles. Fiber area was determined by direct planimetry. The plantaris and soleus muscles hypertrophied 103% and 45%, respectively, within 60 days. Compensatory hypertrophy of the plantaris muscle was accompanied by a significant but disproportionate increase in the cross-sectional areas of the three muscle fiber types. There was an approximate 4-fold increase in the number of slow-twitch-oxidative (SO) fibers observed per transverse section. The hypertrophied plantaris muscle exhibited a significantly greater number of fibers per cross-section (29%) than the respective control muscle. The compensated soleus muscle consisted of nearly 100% SO fibers compared to 83% for the control soleus muscle.     

Temporal progress of muscle adaptation to endurance training in hind limb muscles of young rats

Summary The soleus, rectus femoris and gastrocnemius muscles of young rats were studied after 3, 6 and 12 weeks of treadmill training. The muscle fibers were characterized histochemically by their succinate dehydrogenase (SDH) and myofibrillar ATPase activity, and morphometrically by their cross-sectional areas, which were corrected for different body weights of control and trained animals.After 12 weeks of training the mean area of fibers in the muscles studied was not significantly different from the controls, as expected. In the soleus muscle the percentage of the fast-twitch fibers was decreased as a result of their transformation into slow-twitch fibers. Trained soleus muscles were the only muscles showing pathologically altered fibers, suggesting overload. The percentages of fiber types and their areas exhibited changes specific for the muscles and muscle regions studied.From these results it is concluded that the adaptation follows the sequence proportional adaptation of morphometrical parameters, disproportional adaptation of the areas of fiber types, and disproportional adaptation of the percentages and/or the areas of the fiber types. It is shown by comparison with the literature that this sequence may be generalized to a sequence of increasing expense necessary for the adaptation to increasing stimuli, and that the most decisive factors for adaptation are work load, frequency of exercise, period of training, and the age of the subject at the initiation of the training.