Diabetic state-induced activation of calcium-activated neutral proteinase in mouse skeletal muscle

The effect of a diabetic state in the diabetic KK-CAy mouse on calcium activated neutral proteinase (CANP) of hind-limb skeletal muscles was investigated. In the diabetic state, there was an increased sensitivity to activation of CANP by calcium (Ca). In addition, there was an enhancement of maximal activity of the enzyme. The effect was induced by secondary modification of the diabetic state, but not genetical factors. Several lines of evidence suggest that the CANP is responsible for 92 K dalton protein in diabetic skeletal muscles. Among the evidence are the following: a) The 92 K band in the diabetic muscles was lower than in the prediabetic mouse and restored by the addition of 2 mM EDTA and 2 mM EGTA. b) The band was reduced by increasing the Ca content and neutral pH in the non-diabetic normal muscles. c) E-64-C, a CANP inhibitor, restored the 92 K component reduced by the diabetic state. Since the band in denervated muscles was not changed by the Ca chelating agents, the reduction of the band in the diabetic muscles is related with musculotrophic factors, not diabetic neuropathy. These results suggest that diabetic amyotrophy may be regarded as a phenomenon linked to an increase in intracellular Ca ions and an increase in CANP activity.     

Diabetic state-induced modification of Ca, Mg, Fe and Zn content of skeletal, cardiac and smooth muscles

The metal content of diaphragm, gastrocnemius, ventricle, and bladder muscles in genetically obese diabetic KK-CAy and alloxan-diabetic ddY mice was compared with that in prediabetic KK-CAy and normal ddY mice, because the muscles of the diabetic KK-CAy mice had morphological abnormalities, such as atrophy, disappearance of Z-band, disturbed myofibrils and swollen sarcoplasmic reticulum. The amounts of calcium (Ca) in gastrocnemius, ventricle and bladder muscles from the prediabetic KK-CAy mice were significantly 7.7, 98.3, and 36.9% greater, respectively, than those in normal ddY mice. In contrast, the magnesium (Mg) content of the diaphragm, the gastrocnemius, and the ventricle in the prediabetic mice was 8.6, 7.4, and 4.3% lower, respectively, than in the ddY mice. The iron (Fe) content of the diaphragm, gastrocnemius, and ventricle muscles in the prediabetic mice was 29.2, 43.6, and 44.6% greater, respectively, than in the ddY mice. The Ca content in the gastrocnemius muscles of the diabetic KK-CAy mice and the alloxan-diabetic mice was 19.8 and 11.7% higher, respectively, than in the prediabetic and normal mice. The Ca content of the ventricle muscle was increased only in the alloxan-mice. The gastrocnemius Mg was also 9.0 and 5.5% greater in the KK-CAy and the alloxan-mice. The Fe content of the diaphragm and the gastrocnemius muscles from the KK-CAy mice was 27.3 and 23.2% greater, respectively, than in the prediabetic mice. The zinc (Zn) content of the gastrocnemius and the bladder was 16.4 and 18.0% higher, but the ventricle Zn was 13.4% lower, respectively, than in the prediabetic control. The changes in metal content induced by the diabetic state may be related to the morphological abnormalities.     

Dissociation between insulin binding and glucose utilization after intense exercise in mouse skeletal muscles

Since there are data to indicate that heavy exercise decreases insulin binding to skeletal muscle at a point when glucose uptake is known to be augmented, we tested the hypothesis that insulin-stimulated glucose uptake and metabolism are dissociated from insulin binding after exercise. Therefore, insulin binding, 2-deoxy-d-glucose (2-DOG) uptake and glucose incorporation into glycogen and glycolysis were compared in soleus and EDL muscles of intensively exercised (2-3 h) mice and non-exercised mice. Basal 2-DOG uptake was increased in the exercised EDL (P less than 0.05) but not in the exercised soleus (P greater than 0.05). However, in both muscles intense exercise increased insulin-stimulated (0.1-16 nM) 2-DOG uptake (P less than 0.05). The rates of glycogenesis were increased in both the exercised muscles (P less than 0.05) as was the rate of glycolysis in the exercise soleus (P less than 0.05). Glycolysis was not altered in the EDL (P greater than 0.05). In the face of the increased 2-DOG uptake and glucose metabolism in the exercised muscles, insulin binding was not altered in the exercised soleus muscle (P greater than 0.05) and was decreased in the exercised EDL (P less than 0.05). These results indicate that after intense exercise there is a dissociation of insulin binding from insulin action on glucose uptake and metabolism in skeletal muscles.     

Solubilization and reconstruction of microsomal AMP-deaminase from skeletal muscles]

Microsomal AMP-deaminase was solubilized by 0.5 M KCl after treatment of microsomal membranes with 0.12 M KCl. Using disc-electrophoresis in polyacrylamide gel in the presence of sodium dodecyl sulfate one major protein component (mol. weight about 90 000) and three minor ones with molecular weights of 110 000, 80 000, and 60 000 were found in the soluble fraction. In addition to proteins, the fraction was found in the soluble fraction. In addition to proteins, the fraction was found to contain a small amount of phospholipids. The deaminase found in the solution may be reconstructed into the membranes at a decrease in KCl concentration, part of enzyme being bound in the inactive form under excess of the soluble fraction. Deaminase binding to the membranes is unaffected by the changes within the pH range of 6.2--7.8 and temperature range of 4--10 degrees C. It is assumed that AMP-deaminase is bound to other membrane components by electrostatic bonds.     

Comparative biochemical study of sarcolemma and sarcoplasmic reticulum fractions isolated from mouse skeletal and cardiac muscles

1. Ca-ATPase activity, calcium-binding proteins and Concanavalin-A-bound glycoproteins of sarcolemma and sarcoplasmic reticulum were compared in mouse cardiac and skeletal muscles. 2. Ca-ATPase activity and calsequestrin were quite reduced in cardiac muscle, and the quantity of calcium bound to these two proteins was practically negligible, contrary to what was observed with skeletal muscle. In addition, the quantity of lipid bound calcium was not greater in cardiac muscle than in skeletal muscle. 3. Certain proteins seemed exclusively specific for skeletal muscle, including a 30,000 mol. wt glycoprotein which was totally absent in cardiac muscle sarcolemma.     

The in vitro effect of corticosterone on insulin binding and glucose metabolism in mouse skeletal muscles

We studied the in vitro effect of corticosterone on insulin binding, uptake of 2-deoxy-D-glucose, glycolysis, and glycogenesis in the soleus and extensor digitorum longus (EDL) of Swiss-Webster mice. In each experiment, one muscle (soleus/EDL) was incubated with corticosterone (0.1, 1, 50, and 100 micrograms/mL) and the respective contralateral muscle was incubated without corticosterone, but at the same insulin and pH levels. Corticosterone did not affect insulin binding in both muscles. However, corticosterone decreased the uptake of 2-deoxy-D-glucose and the rate of glycolysis and glycogenesis in both muscles when the dose was pharmacologic (50 and 100 micrograms/mL), but not when it was physiologic (0.1 and 1 microgram/mL). For glycolysis and glycogenesis, the suppression was greater in the EDL when compared with the soleus. This suppression was seen in both basal and insulin-stimulated conditions. In this in vitro system, where the experimental muscle is not exposed to prior hyperinsulinemia as in the in vivo model, corticosterone, at pharmacologic doses, affects postreceptor events without altering the insulin binding in the skeletal muscle.     

Fine structure of myomyous junctions in the mouse skeletal muscles

The reaction product of acetylcholinesterase (AChE) activity is known to be specifically localized at a neuromuscular junction and a muscle-tendon junction of the striated skeletal muscles. In addition to the two junctions, we recently found some linear precipitates due to AChE activity running transversely across a fibre of the semitendinosus, rectus abdominis, gastrocnemius, tibialis anterior and diaphragm muscles in mice. Under an electron microscope, the linear precipitates were seen at the extracellular side of the muscle fibre endings. Most of the endings contacted each other to form a junction, which has been called the 'myomyous junction (M-Mj)'. The patterns of the M-Mj were grouped into three types: (1) a junction in which all contacts were firm, without any connective tissue, and invaginated deeply; (2) the ones in which numerous collagen fibres were visible in the space between the two separate opposing muscle fibres; (3) an intermediate type between (1) and (2), i.e. a junction with partial contacts. The muscle fibre ending forming M-Mj was constructed of finger-like processes like that of a muscle-tendon junction. However, the processes of a M-Mj adhered so closely to each other that no collagen fibrils could penetrate into their folds.     

Temporally controlled targeted somatic mutagenesis in skeletal muscles of the mouse

To generate temporally controlled targeted somatic mutations selectively and efficiently in skeletal muscles, we established a transgenic HSA-Cre-ER(T2) mouse line in which the expression of the tamoxifen-dependent Cre-ER(T2) recombinase is under the control of a large genomic DNA segment of the human skeletal muscle alpha-actin gene, contained in a P1-derived artificial chromosome. In this transgenic line Cre-ER(T2) is selectively expressed in skeletal muscles, and Cre-ER(T2)-mediated alteration of LoxP flanked (floxed) target genes is skeletal muscle-specific and strictly tamoxifen-dependent. HSA-Cre-ER(T2) mice should be of great value to analyze gene function in skeletal muscles, and to establish animal models of human skeletal muscle disorders.     

T-system membranes in microsomal fractions of crayfish muscles: identification and differences in protein composition

Specific binding of 3H-ouabain and ruthenium red (RR) to membranes of T-tubules in crayfish muscles was used to identify the subfraction containing vesicles originating from the T-system. The microsomal fraction was prepared by differential centrifugation, and subfractions were separated in continuous or discontinuous sucrose density gradients. 3H-ouabain binding was estimated by scintillation counting; RR binding was examined by electron microscopy. The light subfraction was identified using both methods as that containing vesicles of T-tubules. Protein separation by SDS-electrophoresis revealed marked differences between the subfraction containing vesicles of T-tubules and other subfractions, the most distinctive feature being the presence of a protein of Mr 46,000 predominantly in the light subfraction.     

Endurance training decreases the alkaline proteolytic activity in mouse skeletal muscles

Alkaline and myofibrillar protease activities of rectus femoris, soleus, and tibialis anterior muscles and the pooled sample of gastrocnemius and plantaris muscles were analyzed in male NMRI-mice during a running-training program of 3, 10, or 20 daily 1-h sessions. The activity of citrate synthase increased during the endurance training, reflecting the increased oxidative capacity of skeletal muscles. The activities of alkaline and myofibrillar proteases continually decreased in the course of the training program in all muscles studied. Instead, the activity of beta-glucuronidase (a marker of lysosomal hydrolases) increased in all muscles. The highest activities were observed at the beginning of the training program. Present results, together with our earlier observations, show that the type of training, running as opposed to swimming, modulates the training responses in alkaline protease activities. Further, diverse adaptations in the activities of alkaline proteases and a lysosomal hydrolase suggest difference in the function of different proteolytic systems.     

Experimentally induced binding of phytochrome to mitochondrial and microsomal fractions in etiolated pea shoots

Summary A brief irradiation with red light of pea (Pisum sativum L.) shoot segments kept at 0° resulted in very rapid binding of both Pr and Pfr to mitochondrial and microsomal fractions. The effect was not far-red reversible. The amount of phytochrome bound to the mitochondrial fraction was proportional to the percentage of Pfr of the fraction, and the ratio of Pr and Pfr in the bound form was the same as that in 12,000 x g supernatant. After a brief exposure of the segments to red light at 0° and a subsequent dark incubation at 30° in Tris-HCL buffer containing dithiothreitol or EDTA, which bot inhibit Pfr decay, the contents of phytochrome in the mitochondrial and microsomal fractions were significantly enhanced with time. The red-light effect was reversed by far-red light. The increase of the phytochrome content in the particulate fractions continued for at least 2 h, reaching a ca. 3 times higher level in terms of (A) per mg protein.Abbreviations R red - FR far-red - Pr red-absorbing form of phytochrome - Pfr far-red-absorbing form of phytochrome     

Androgen and estrogen binding in rat skeletal and perineal muscles.

Specific in vitro binding of [3H]testosterone (T), 5ALPHA[3H]dihydrotestosterone (DHT), and [3H[estradiol (E2) was demonstrated in the 30 000 X g supernatant (cytosol) of thigh muscles (TM) and of the levator ani - bulbocavernosus muscle complex (LA-BC) by gel filtration through Sephadex G-25 columns. In TM cytosol, T and E2 [are bound with high affinity (Ka = 1.1 X 10(9) M-1, and 2.3 X 10(9) M-1 respectively) whereas DHT binding is of lower affinity (Ka = 5.0 X 10(7) M-1).] In LA-BC cytosol, T, E2, and DHT are bound with high affinity (Ka = 1.9 X 10(9) M-1, 0.3 X 10(9) M-1, and 0.5 X 10(9) M-1, respectively). Competition experiments suggest that the binding of the three hormones (T, E2, and DHT) is due to different proteins. In addition to TM and LA-BC, T and E2 binding was found in other muscles of male and female rats, including gastrocnemius, the pectoralis, diaphragm, and heart.