Glycogen synthase kinase-3 is the predominant insulin-regulated eukaryotic initiation factor 2B kinase in skeletal muscle

Eukaryotic initiation factor eIF2B is a guanine nucleotide exchange protein involved in regulation of translation initiation. Phosphorylation of the epsilon-subunit is thought to be important in insulin-mediated changes in eIF2B activity. However, elucidation of insulin's action has proven elusive, primarily because eIF2B epsilon is a substrate in vitro for at least three different protein kinases. In the present study, we observed changes in eIF2B epsilon kinase activity only in those muscles previously shown to exhibit alterations in protein synthesis in response to insulin. Specifically, eIF2B epsilon kinase activity was increased in psoas muscle from diabetic rats compared to controls. Treating diabetic rats with insulin rapidly reduced eIF2B epsilon kinase activity below control values. Changes were not observed in heart. To identify the kinase(s) in psoas responsible for phosphorylating eIF2B epsilon, the wildtype and two variant forms of the epsilon-subunit were expressed in and purified from Sf9 insect cells, and were used as substrates in protein kinase assays. The first variant contained a point mutation in the eIF2B epsilon cDNA that converted the glycogen synthase kinase-3 (GSK-3) phosphorylation site, Ser535, to a nonphosphorylatable Ala residue. In the second variant, the putative GSK-3 'priming' site, Ser539, was converted to Asp. Based on the pattern of phosphorylation of the wildtype and two variant forms of eIF2B epsilon using casein kinase (CK)-I, CK-II, or GSK-3 as well as that observed with skeletal muscle extracts, we conclude that the predominant eIF2B epsilon kinase in psoas muscle is GSK-3. Thus, insulin-mediated changes in eIF2B activity are likely to involve GSK-3.     

Effects of starvation, diabetes and acute insulin treatment on the regulation of polypeptide-chain initiation in rat skeletal muscle.

The rate of protein synthesis in skeletal muscle is greatly decreased in response to diabetes and starvation. Analysis of polyribosome profiles indicates that polypeptide-chain initiation is impaired under these conditions. To identify the step in initiation that is affected, we assayed the incorporation of [35S]methionyl-tRNAfMet into [35S]methionyl-tRNAfMet . 40S-ribosomal-subunit initiation complexes in cell-free extracts based on postmitochondrial supernatants prepared from gastrocnemius muscle. Extracts from either starved or diabetic rats were 30-40% less active in forming these complexes compared with those derived from fed or insulin-maintained controls respectively. This change could be reversed by treatment of either starved or diabetic rats with insulin in vivo 30 min before death. Formation of 40S initiation complexes by extracts from either fed or starved rats could be stimulated by the addition of exogenous purified initiation factor eIF-2, but extracts from starved or diabetic rats were more sensitive than controls to stimulation by low concentrations of the factor. These results provide evidence for the acute regulation by insulin of protein synthesis in skeletal muscle at the level of polypeptide-chain initiation, and suggest that in this tissue, as in certain other eukaryotic systems, control of initiation appears to be mediated by changes in the activity of initiation factor eIF-2.     

Dosage effect of streptozotocin on rat tissue enzyme activities and glycogen concentration

The effect of different dosages of streptozotocin (STZ) on selected rat tissue enzyme activities and glycogen concentration were investigated. The rats were administered STZ intravenously at 60 (STZ-60), 80 (STZ-80), 100 (STZ-100), and 150 (STZ-150) mg/kg body weight. They were used 3 weeks postinjection. Mortality prior to kill occurred only in the STZ-100 and STZ-150 rats. All diabetic rats showed reduced growth rate, hyperglycemia, hypoinsulinemia, and hyperlipemia. Phosphofructokinase (PFK) and succinate dehydrogenase (SDH) activities were significantly reduced in the red gastrocnemius muscle of all diabetic rats, and in the white gastrocnemius and soleus of STZ-100 and STZ-150 groups. PFK activity in the heart remained unaltered, but SDH activity was below normal. Liver SDH activity was not affected by insulin deficiency. Glycogen content was markedly increased in the heart and decreased in the liver of all diabetic rats. Glycogen content in the skeletal muscle was similar to the controls, except for the lower values in the soleus of STZ-100 and STZ-150 rats. When STZ-80 and STZ-150 rats were given insulin therapy, the STZ-80 rats showed a greater response to the treatment. Despite similar levels of plasma immunoreactive insulin among all groups of diabetic rats, the STZ-100 and STZ-150 rats had higher mortality, greater loss in body weight, and alterations in enzyme activities and glycogen content in the tissues studied.     

UCP3 gene expression does not correlate with muscle oxidation rates in troglitazone-treated Zucker fatty rats

Uncoupling protein-3 (UCP3), a mitochondrial carrier protein predominantly expressed in muscle, has been suggested to release stored energy as heat. The insulin-sensitizing thiazolidinediones enhance glucose disposal in skeletal muscle and have been reported to increase the expression of uncoupling proteins in various experimental systems. We therefore studied the effect of troglitazone treatment on UCP3 gene expression in muscles from lean and obese Zucker rats. In comparison with obese littermates, basal UCP3 mRNA levels in lean Zucker rats tended to be higher in white and red gastrocnemius muscles, but were lower in soleus (P<0.001) muscle and heart (P<0.01). In lean rats, troglitazone significantly increased UCP3 gene expression in white and red gastrocnemius and heart muscles (all P<0.01). In contrast, the drug reduced UCP3 mRNA expression in red gastrocnemius and soleus muscles of obese littermates (all P<0.001). The troglitazone-dependent decrease in UCP3 gene expression was accompanied by an increased weight gain in obese rats, while no such effect was observed in lean rats. In obese rats, improvement of insulin resistance by troglitazone was associated with increased rates of basal and insulin-stimulated CO(2) production from glucose measured in soleus muscle. These studies demonstrate that effects of troglitazone on UCP3 gene expression depend on the phenotype of Zucker rats and that troglitazone-induced metabolic improvements are not related to increased uncoupling resulting from upregulation of UCP3 mRNA expression in muscle.     

Enzyme activities and reduced insulin need with training in streptozotocin-induced diabetic rats

Following a 12-week endurance training program, the SDH activities of gastrocnemius medialis muscle of streptozotocin-induced diabetic animals increased by 50%. On the other hand, a 14% decrease was observed in the same muscle of diabetic animals submitted to power training. No difference between groups, for soleus and gastrocnemius lateralis muscles following the two different training regimens was observed. It can be concluded that streptozotocin-induced diabetic animals controlled by daily insulin demonstrate a normal muscle enzyme adaptation to exercise. These data also demonstrate that regular power and/or endurance exercise can result in reduced exogenous insulin need in streptozotocin-induced diabetic rats (18% and 28% respectively), suggesting a more efficient membrane transport of glucose with induced exercise, and a decreased need for insulin supported transport.     

Tissue-specific regulation of protein synthesis by insulin and free fatty acids

The purpose of the study described herein was to investigate how the mammalian target of rapamycin (mTOR)-signaling pathway and eukaryotic initiation factor 2B (eIF2B) activity, both having key roles in the translational control of protein synthesis in skeletal muscle, are regulated in cardiac muscle of rats in response to two different models of altered free fatty acid (FFA) and insulin availability. Protein synthetic rates were reduced in both gastrocnemius and heart of 3-day diabetic rats. The reduction was associated with diminished mTOR-mediated signaling and eIF2B activity in the gastrocnemius but only with diminished mTOR signaling in the heart. In response to the combination of acute hypoinsulinemia and hypolipidemia induced by administration of niacin, protein synthetic rates were also diminished in both gastrocnemius and heart. The niacin-induced changes were associated with diminished mTOR signaling and eIF2B activity in the heart but only with decreased mTOR signaling in the gastrocnemius. In the heart, mTOR signaling and eIF2B activity correlated with cellular energy status and/or redox potential. Thus FFAs may contribute to the translational control of protein synthesis in the heart but not in the gastrocnemius. In contrast, insulin, but not FFAs, is required for the maintenance of protein synthesis in the gastrocnemius.     

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.     

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.     

Evidence for increased peroxidative activity in muscles from streptozotocin-diabetic rats

The ability of cardiac and skeletal muscles from diabetic rats to metabolize superoxide and hydrogen peroxide was determined by the activities of superoxide dismutase (SOD) and catalase, respectively. Male and female Sprague-Dawley rats, 43 days old, were made diabetic with a single intravenous injection of streptozotocin (70 mg/kg body weight). On the 80th day after injection the blood glucose concentration of these rats was increased fourfold, and the plasma insulin concentration was decreased four- to fivefold compared to controls. Body weights of male diabetic rats were 61% and those of female diabetic rats were 66% of their ad libitum-fed controls. The seven different skeletal muscles examined weighed less in the diabetic rats than in controls of the same age and body weight. The hearts of the diabetic rats weighed more than those of controls of the same age and body weight. Comparison to the body weight controls allowed the distinction of specific effects due to lack of insulin from effects due to retardation in muscle growth. Increased catalase activity in all muscles examined from diabetic rats (plantaris, gastrocnemius, and heart) suggested a response in catalase activity similar to that of starved rats. SOD activity was not altered in the diabetic rat skeletal muscles and erythrocytes, but was somewhat decreased in the heart.     

The significance of lipoprotein lipase in rat skeletal muscles.

Lipoprotein lipase was assayed in extracts of acetone-ether powders of rat skeletal muscles. Enzyme activity in soleus had typical characteristics of lipoprotein lipase in other tissues: inhibition by molar NaCl and protamine sulfate and activation by the human apolipoprotein, R-glutamic acid. Activity in muscles with predominantly red fibers (soleus, diaphragm, lateral head of gastrocnemius and anterior band of semitendinosus) was higher than in those with predominantly white fibers (body of gastrocnemius and posterior band of semitendinosus). No effect of a 24 hour fast upon enzyme activity was observed in ten skeletal muscles, but activity decreased substantially in four adipose tissue depots and increased slightly in heart muscle with fasting. Four minutes after intravenous injection of labeled lymph chylomicrons, skeletal muscles with predominantly red fibers incorporated several times more chylomicron triglyceride fatty acids than thos with predominantly white fibers. Estimated lipoprotein lipase activity in total skeletal muscle was about two-thirds that in total adipose tissue of rats fed ad libitum. After a 24 hour fast, total activity in skeletal muscle was about twice that in adipose tissue. These data suggest that a substantial fraction of lipoprotein lipase is in skeletal muscle of rats and that this tissue, especially its red fibers, is an important site of removal of triglycerides from the blood.     

Studies on the utilization and mobilization of lipid in skeletal muscles from streptozotocin-diabetic and control rats.

Several aspects of lipid metabolism in the soleus and diaphragm muscles of streptozotocin-diabetic and control rats were investigated. The triglyceride content of both muscles was elevated in the diabetic state and the presence of increased intracellular lipid was confirmed by electron microscopy. In vitro glucose and palmitate oxidation studies showed that both types of muscle from the diabetic animals metabolized more fat than did the soleus and diaphragm from control rats. While isoproterenol alone produced a significant lipolytic response in both the soleus and diaphragm from control and diabetic animals, there was no difference in the percent increase in fatty acids released from muscles of diabetic rats compared to controls. However, the absolute difference was greater when the diaphragms were compared. Muscles from experimental and control animals showed a marked reduction in the amount of free fatty acids released in response to insulin. In addition, in the presence of the hormone, both the absolute and percent isoproterenol-stimulated increases in fatty acids were significantly greater for both diaphragm and soleus muscles from diabetic rats. The effects of insulin, isoproterenol, and the combination of these two hormones on the amount of glycerol released into the incubation medium were similar to those found on free fatty acid release. The results of these experiments show that there is an apparent increase in fat utilization in skeletal muscle of diabetic rats. Furthermore, measurements of triglyceride concentration and the enhanced response to isoproterenol stimulation in the muscles from these animals suggests that they may have an increased capacity for mobilization of intracellular lipids. Finally, in the diabetic state, both the soleus and diaphragm appear to demonstrate an increased response to the antilipolytic effect of insulin as measured by the decreased amount of fatty acid released into the incubation medium, the percent change also being significant for the soleus muscle.-Stearns, S. B., H. M. Tepperman, and J. Tepperman. Studies on the utilization and mobilization of lipid in skeletal muscles from streptozotocin-diabetic and control rats.     

The effect of three hypolipidemic drugs on catalase activity and peroxisomal and mitochondrial palmitate oxidation in rat cardiac and skeletal muscle

Catalase activity and peroxisomal and mitochondrial palmitate oxidation have been investigated in cardiac and skeletal muscle from rats fed clofibrate, ciprofibrate or nafenopin in an unrefined diet for different periods of time. Nafenopin was also added to either a high carbohydrate (70% of kilocalories from glucose) or high fat (70% of kilocalories from lard) diet and fed to rats for either 1 or 3 weeks. Catalase activity was elevated in all muscles from rats fed the hypolipidemic drugs. The response of catalase activity in muscle to clofibrate was dose-dependent. The response time of catalase activity was different in individual muscles. Peroxisomal palmitate oxidation was elevated in the heart and soleus muscle from rats fed nafenopin in either the high-carbohydrate or the high-fat diet. There was no change in peroxisomal palmitate oxidation in psoas or extensor digitorum longus muscle from rats fed the drugs. Mitochondrial palmitate oxidation was only slightly increased by nafenopin in the heart and soleus muscles after 3 weeks of nafenopin feeding. The results suggest that the cardiac muscle, like the liver, responds to hypolipidemic drug treatment with an increase in peroxisomal fat oxidation. The skeletal muscle response is less specific and that tissue may not contribute to the hypolipidemic effect of the drugs. The findings also suggest that these drugs do not induce peroxisome proliferation in skeletal muscle.     

Protein synthesis in skeletal muscle of the perfused rat hemicorpus compared with rates in the intact animal.

The rate of protein synthesis was measured in muscles of the perfused rat hemicorpus, and values were compared with rates obtained in whole animals. In gastrocnemius muscle of fed rats the rate of synthesis measured in the hemicorpus was the same as that in the whole animal. However, in plantaris, quadriceps and soleus muscles rates were higher in the hemicorpus than those in vivo. In the hemicorpus, starvation for 1 day decreased the rate of protein synthesis in gastrocnemius and plantaris muscles, in parallel with decreases in the RNA content, but the soleus remained unaffected. Similar effects of starvation were observed in vivo, so that the relationships between rates in vivo and in the hemicorpus were the same as those in fed rats. Proteins of quadriceps and plantaris muscles were separated into sarcoplasmic and myofibrillar fractions. The rate of synthesis in the sarcoplasmic fraction of the hemicorpus from fed rats was similar to that in vivo, but synthesis in the myofibrillar fraction was greater. In the plantaris of starved rats the rates of synthesis in both fractions were lower, but the relationships between rates measured in vivo and in the perfused hemicorpus were similar to those seen in fed rats. The addition of insulin to the perfusate of the hemicorpus prepared from 1-day-starved animals increased the rates of protein synthesis per unit of RNA in gastrocnemius and plantaris muscles to values above those seen in fed animals when measured in vivo or in the hemicorpus. Insulin had no effect on the soleus. Overall, the rates of protein synthesis in the hemicorpus differed from those in vivo. However, the effect of starvation when measured in the whole animal was very similar to that measured in the isolated rat hemicorpus when insulin was omitted from the perfusate.     

The effect of glucagon administration on protein synthesis in skeletal muscles, heart and liver in vivo.

Infusion of glucagon (0.5 mg/h per 100 g body wt.) into fed rats for 6 h inhibited protein synthesis in skeletal muscle, but not in heart. The order of sensitivity of three muscles was plantaris greater than gastrocnemius greater than soleus. Treatment with glucagon for periods of 1 h or less had no effect. Liver protein synthesis was inhibited by glucagon treatment for 10 min, but stimulated after 6 h. The effect of glucagon on muscle was not secondary to impaired food absorption or to depletion of amino acids by increased gluconeogenesis, since the inhibition of protein synthesis was observed in postabsorptive and amino acid-infused rats. The failure of glucagon to inhibit muscle protein synthesis after 1 h may have been caused by the increase in plasma insulin that occurred at this time, since an inhibition was detected in insulin-treated diabetic rats. The lowest infusion rate that gave a significant decrease in muscle protein synthesis was 6 micrograms/h per 100 g body wt., despite a small increase in plasma insulin. This gave plasma glucagon concentrations in the high pathophysiological range, suggesting that glucagon may be significant in the pathogenesis of muscle wasting in metabolic stresses such as diabetes and starvation.     

Modulation of muscle insulin resistance by selective inhibition of GSK-3 in Zucker diabetic fatty rats

A role for elevated glycogen synthase kinase-3 (GSK-3) activity in the multifactorial etiology of insulin resistance is now emerging. However, the utility of specific GSK-3 inhibition in modulating insulin resistance of skeletal muscle glucose transport is not yet fully understood. Therefore, we assessed the effects of novel, selective organic inhibitors of GSK-3 (CT-98014 and CT-98023) on glucose transport in insulin-resistant muscles of Zucker diabetic fatty (ZDF) rats. Incubation of type IIb epitrochlearis and type I soleus muscles from ZDF rats with CT-98014 increased glycogen synthase activity (49 and 50%, respectively, P < 0.05) but did not alter basal glucose transport (2-deoxyglucose uptake). In contrast, CT-98014 significantly increased the stimulatory effects of both submaximal and maximal insulin concentrations in epitrochlearis (37 and 24%) and soleus (43 and 26%), and these effects were associated with increased cell-surface GLUT4 protein. Lithium enhanced glycogen synthase activity and both basal and insulin-stimulated glucose transport in muscles from ZDF rats. Acute oral administration (2 x 30 mg/kg) of CT-98023 to ZDF rats caused elevations in GSK-3 inhibitor concentrations in plasma and muscle. The glucose and insulin responses during a subsequent oral glucose tolerance test were reduced by 26 and 34%, respectively, in the GSK-3 inhibitor-treated animals. Thirty minutes after the final GSK-3 inhibitor treatment, insulin-stimulated glucose transport was significantly enhanced in epitrochlearis (57%) and soleus (43%). Two hours after the final treatment, insulin-mediated glucose transport was still significantly elevated (26%) only in the soleus. These results indicate that specific inhibition of GSK-3 enhances insulin action on glucose transport in skeletal muscle of the insulin-resistant ZDF rat. This unique approach may hold promise as a pharmacological treatment against insulin resistance of skeletal muscle glucose disposal.     

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.     

Antioxidant capacity in rat skeletal muscle tissues determined by electron spin resonance

The amount of radical scavenging activity in muscle is unknown. The present study examines whether electron spin resonance (ESR) could measure and distinguish antioxidant capacity in muscle with different contractile and metabolic characteristics. Specimens of the soleus, plantaris, gastrocnemius (deep/surface portions), heart and diaphragm were obtained from female Wistar rats (n=7; 12 weeks old). Scavenging activity against superoxide anions in these specimens were determined by ESR using a spin-trapping chemical (5,5-dimethyl-1-pyrroline-N-oxide). The ESR signal intensity of reaction mixtures containing muscle tissues was significantly lower in the heart, soleus, diaphragm and deep portion of the gastrocnemius than in the plataris and surface portion of the gastrocnemius. Thus, the amount of scavenging activity converted into superoxide dismutase activity was the highest in the heart, and higher in the soleus, diaphragm and deep portion of the gastrocnemius than in other muscles (ANOVA, P<0.01). In addition, scavenging activity significantly correlated with citrate synthase activity (r=0.72, P<0.01, n=42) and myoglobin content (r=0.63, P<0.01, n=42). These findings suggested that ESR and spin-trapping can be detect differences in free radical scavenging activity among muscle tissues with different metabolic characteristics.     

Developmental Changes in Lactate Dehydrogenase and Aldolase Activity of the A2G-adr Mouse with Abnormal Muscle Function: Further Comparison with the 129Re-dy Mutant

Abstract: Lactate dehydrogenase and aldolase activity were reduced in lateral gastrocnemius muscle from two mouse mutants, A2G- adr and 129Re- dy , with abnormal muscle function. The activities of both of these enzymes were significantly reduced in the lateral gastrocnemius muscle from the A2G- adr mice at ages varying from 2 weeks to 32 weeks, whereas the activities in the soleus, heart, liver, and brain were the same as in the control animals. The lactate dehydrogenase isoenzymes in the lateral gastrocnemius and soleus muscles from the A2G mice were quantified, and although those of the soleus were comparable in mutant and control muscle, the lateral gastrocnemius from the adr mutant had reduced activity of LDH 5 and increased activities of the other four isoenzymes. The findings suggest that the adr mutation is expressed in the white (Type II) muscle fibres and not in the red (Type I) fibres or in any of the organs studied. It is suggested that the initiation of differentiation into Type II fibres from the embryonic form is absent or delayed in the A2G mutant. The reduced activities of lactate dehydrogenase and aldolase in 129Re- dy muscle confirm the findings of other workers.     

Decreased lysosomal protease content of skeletal muscles from streptozotocin-induced diabetic rats: a biochemical and histochemical study

Summary The activity of four lysosomal proteases in soleus and extensor digitorum longus muscles was studied in streptozotocin-induced diabetic rats using newly developed fluorescence histochemical and biochemical techniques. The results indicate that the content of lysosomal protease in skeletal muscle cells was decreased three weeks after the induction of diabetes. The reduction was most pronounced in the extensor digitorum longus for all the proteases tested, but in the soleus only cathepsin B and dipeptidyl peptidase II showed a decrease. Biochemical assays on total muscle homogenates and muscle extracts confirmed the histochemical observations that protease activity was significantly lower in diabetic muscles. This decrease in activity varied with the duration of diabetes beginning as early as 48 h for the soleus. In conclusion, myofibre-specific decreases in lysosomal proteases occur following diabetes.