Nonreceptor-mediated responses of adenylate cyclase in membranes from liver, muscle, and white and brown adipose tissue of obese (fa/fa) and lean (Fa/) Zucker rats

Adenylate cyclase activity was determined in membranes of liver, muscle, white adipose tissue, and brown adipose tissue (BAT) of lean (Fa/) and obese (fa/fa) Zucker rats. Responses were monitored following beta-adrenergic receptor stimulation and addition of GTP, GTP gamma S, or forskolin. beta-Adrenergic responses in liver, white adipose tissue, and BAT were lower in obese than in lean animals. No such difference was observed in muscle membranes. Production of cAMP after addition of guanine nucleotides was lower in liver and white adipose tissue membranes from obese rats compared with their lean littermates. Synthesis of cAMP in muscle membranes of obese animals after addition of GTP was either not different, or slightly higher, than that observed in muscle membranes from lean animals. Furthermore, production of cAMP after forskolin addition to muscle membranes of obese rats was significantly higher than that observed from lean rats under the same conditions. Interestingly, BAT membranes of obese rats were significantly more sensitive to guanine nucleotide activation than those of lean animals. The results confirm recent findings indicating inferior function of G proteins in liver plasma membranes of obese Zucker rats, and extend this observation to adipose tissue. The present results further suggest that the "nonreceptor" components (e.g., G proteins) responsible for the activation of adenylate cyclase in BAT membranes of obese rats are more responsive to stimulation than those of lean animals. Such sensitivity may be related to and perhaps compensate for the reduced thermogenic activity in the obese Zucker rat during the development of obesity.     

Regulation of lipoprotein lipase in muscle and adipose tissue during exercise.

Lipoprotein lipase (LPL) is regulated in a tissue-specific manner; exercise increases LPL activity in muscle at the same time it is reduced in adipose tissue. The purpose of this study was to determine the relationship between LPL activity and LPL mRNA in muscle and adipose tissue in rats exposed to one bout of exercise. Immediately after a 2-h swim, LPL activity [pmol free fatty acids (FFA).min-1.mg tissue-1] in the exercised animals was reduced 43% in adipose tissue (110 +/- 26 to 63 +/- 17) and increased almost twofold in the soleus muscle (203 +/- 26 to 383 +/- 59) compared with sedentary control animals. At the same time, LPL mRNA was reduced 42% in adipose tissue and increased 50 and 100% in the red vastus and white vastus muscles, respectively. Twenty-four hours after the swim, LPL activity had returned to control levels in adipose tissue and the soleus muscle. At hour 24 of recovery, LPL mRNA was still reduced 23% in the adipose tissue of exercised animals but was not significantly different between exercised and control animals in any of the muscle tissues analyzed. Changes in total RNA concentration could not account for the changes in relative LPL mRNA expression. The relationship between LPL enzyme activity and LPL mRNA in muscle and adipose tissue was +0.86 and +0.93 at 0 and 24 h postexercise, respectively. Thus the tissue-specific changes in enzyme activity induced by exercise could be mediated, in part, through pretranslational control.     

Radioglucose metabolism by richardson's ground squirrels in the weight-gain and weight-loss phases of the circannual cycle

Summary Captive fed, starved, and refed Richardson's ground squirrels in the weight-gain and weight-loss phases of the circannual cycle were injected with radioglucose and the activity of the label in skeletal muscle proteins and white adipose tissue lipids four hours after injection was used to determine if lean body mass and white adipose tissue would be rapidly restored when starved animals were refed. Starvation for six days reduced carcass mass 27–31% and white adipose tissue mass 23–24% (Table 1). Activity of the label in both tissues of weight-gain and weight-loss animals was reduced by starvation. After four days of refeeding activities retured to levels similar to those in fed animals, with the exception of lower activity in skeletal muscle proteins of weight-gain animals. Furthermore, activity in each tissue fraction of starved and refed weight-gain animals was similar to that in weight-loss animals when expressed as per cent of activity in the respective fed state (Table 2). Radioglucose incorporation indicated that when skeletal muscle and adipose tissue are depleted by starvation, distribution of the label upon refeeding is similar to that in the fed state. Four days after refeeding weight-gain phase ground squirrels had restored 5.5 g of lean body mass and 7.5 g of adipose tissue, including 1.4 g (6 kcal) of protein and 7.0 g (66 kcal) of lipid, respectively. These results are also consistent with the fed state, in which weight-gain animals were depositing more lipid than lean body mass.     

Maximal activities of enzymes involved in adenosine metabolism in muscle and adipose tissue of rats under conditions of variations in insulin sensitivity

The maximal activities of 5'-nucleotidase, adenosine deaminase and adenosine kinase were measured in quadriceps or soleus muscle from animals in which the sensitivity to insulin was changed. Most conditions caused no effect on the activities but exercise-training increased the activity of adenosine deaminase and cold exposure increased the activity of 5'-nucleotidase in soleus muscle: in addition, ageing decreased markedly the activities of all three enzymes in both muscles. When the activities are based on mg protein they are much higher in both white and brown adipose tissue than in muscle, suggesting that changes in adenosine concentration may be important in changing insulin sensitivity in adipose tissue whereas changes in adenosine receptor number may be more important in muscle.     

Evidence for defective transfer ribonucleic acid in polymyopathic hamsters and its inhibitory effect on protein synthesis

1. Different reaction steps involved in protein synthesis were studied in skeletal muscles from control and myopathic hamsters. 2. There was no difference between partially purified aminoacyl-tRNA synthetases from myopathic and control animals in yield or catalytic activity, as tested with exogenous deacylated tRNA. 3. However, isolated deacylated tRNA from myopathic muscle was aminoacylated by these synthetases to a lesser extent than that derived from control muscle. 4. Addition of deacylated tRNA isolated from control muscle improved the performance of pH5 enzymes from myopathic muscle in polypeptide synthesis on homologous polyribosomes; tRNA isolated from myopathic animals did not. 5. Preparation of extracts from both types of animals in the presence of the ribonuclease-absorbent bentonite led to an increased capacity of endogenous tRNA to accept amino acids in pH5 enzymes prepared from normal and abnormal tissue, but the difference between the two systems remained the same. 6. Total tRNA nucleotidyltransferase activity, tested with twice-pyrophosphorolysed rat liver tRNA, was identical in both extracts. 7. Added tRNA nucleotidyltransferase incorporated more AMP and CMP into endogenous tRNA with the pH5 enzyme from myopathic muscle than with that from control muscle. 8. Preincubation of deacylated tRNA from myopathic muscle with ATP, CTP and tRNA nucleotidyltransferase more than doubled its subsequent aminoacyl-acceptor activity, and halved the extent of the defect relative to aminoacylation of control tRNA similarly treated. Endogenous tRNA in pH5 enzyme preparations behaved likewise. 9. It is suggested that a 3'-exonuclease in myopathic muscles attacks tRNA molecules in such a way that some of them remain substrates for tRNA nucleotidyltransferase, which may incorporate into RNA not only AMP and CMP, but also GMP. 10. Cell-free protein synthesis in preparations from myopathic hamster muscles is limited by the supply of intact tRNA molecules.     

Ontogeny and kinetics of carnitine palmitoyltransferase in liver and skeletal muscle of the domestic felid (Felis domestica)

The ontogeny of carnitine palmitoyltransferase (CPT) was examined in liver and muscle throughout growth and development of the domestic felid. Homogenates from animals in six age categories (newborn, 24-h, 3-, 6- and 9-week-old, and adult) were examined. Hepatic CPT specific activity increased progressively from birth to 6 weeks and then declined slightly into adulthood, with maximal values for animals greater than 24 h of age [171 nmol/(min g wet tissue)] being 70% higher than for newborns [99 nmol/(min g wet tissue)] (P<.05). Specific activity in adults was similar to that in 6- and 9-week-old juveniles. Total hepatic CPT activity [nmol/(min liver)] increased linearly with age, but the activity expressed per kg body weight [nmol/(min kg BW)] declined after 3 weeks. In contrast, skeletal muscle CPT-specific activity remained unchanged from birth to 3 weeks and then increased significantly, with maximal values at 9 weeks being 90% greater than those for young animals (newborn to 3 weeks; P<.05), whereas specific activity in adults was 50% lower than that observed in 9-week-old animals (P<.05). Hepatic and muscle apparent Km's for carnitine averaged 440 microM and did not vary with age. Hepatic carnitine concentrations remained relatively constant during development, but were lower in adult lactating females, whereas skeletal muscle concentrations increased markedly with age. Hepatic concentrations were 20-50% higher than apparent Km's for carnitine in young and growing animals, but concentrations were similar to the apparent Km at 6 weeks and significantly lower than the apparent Km in adults. Carnitine concentrations in skeletal muscle were 37% lower than apparent Km during the neonatal period, but significantly higher in cats >3 weeks of age. We conclude that postnatal increases in CPT activity support increased capacity for fatty acid oxidation in the developing felid and that dietary carnitine may be required to maximize enzyme activity.     

Cell-free protein synthesis in heart and skeletal muscles from polymyopathic hamsters

1. Cell-free protein synthesis was studied in striated and smooth muscles in an attempt to elucidate the primary genetic defect in polymyopathic hamsters. 2. When washed membrane-free polyribosomes from myopathic and control heart muscle were individually recombined with pH5 enzymes from both types of animals, the pH5 enzymes from myopathic muscle were less active in polypeptide synthesis than those from controls, irrespective of the source of polyribosomes. 3. The same defect was present in skeletal-muscle preparations. 4. Both the initial rate and the maximum extent of incorporation were affected in the defective preparations from myopathic muscle. 5. Concentration differences, with respect to total protein and RNA, were not responsible. 6. Preincubation of the pH5 enzymes resulted in a greater degree of inhibition. 7. The defect in the pH5 enzymes from myopathic muscle was also expressed in poly(U)-directed polyphenylalanine synthesis. 8. Acid proteinase activity in extracts of control and myopathic muscle was the same but general ribonuclease activity in the latter extracts was higher. 9. The defect was also present when both types of pH5 enzymes were prepared in the presence of the ribonuclease-asborbent bentonite. 10. pH5 enzymes from uterine smooth muscle, brains and livers of myopathic animals were similarly affected in homologous and heterologous combinations. 11. It is concluded that the general tissue defect is both qualitative and quantitative in nature, implying that there is a shortage of some essential soluble component in the pH5 fraction which is accompanied by the presence of an altered substituent. This prevents the attainment of extents of polypeptide synthesis in vitro obtained in control extracts from unaffected animals.     

A comparative study of protein synthesis in vitro by skeletal muscle ribosomes from livestock

Skeletal muscle was dissected from the forelimbs of animals and stored at -80 degrees C. Protein synthesis activity was determined in cell-free systems. Incorporation of amino acids into protein was expressed per milligramme of ribosomal RNA, per gramme wet weight of skeletal muscle and per milligramme of DNA. Species related differences in activity were observed in skeletal muscle obtained from pig, horse, lamb, sheep, calf and young steer. Intraspecies differences were seen in cattle of different breeds. The protein composition of the muscle tissue showed quantitative differences between the species. Using biopsy specimens, the system could be used to monitor the protein nutrition status of animals and the production of animal proteins.     

Effect of dinitrosyl iron complexes with thiol-containing ligands on the penile cavernosum bodies in rats

A beneficial effect of dinitrosyl iron complexes (DNIC) with thiol-containing ligands on penile cavernus tissue was shown in rats subjected to penile denervation. Histological and histochemical investigations demonstrated that intracavernous injections of dinitrosyl iron complexes (2 times per one week during 6 months) blocked the reinforcement of endothelial cell proliferation in the tissue characteristic of the cavernous tissue when the penile nerve was removed. On the other hand, treatment with dinitrosyl iron complexes led to the preservation of mitotic activity of smooth myocytes and protected against the appearance in these cells of collagenase, an indicator of muscle transformation into fibrous tissue. It was shown that the process of fibrous transformation of myocytes correlates with a decrease in the mitotic activity of fibroblasts in the adventive part of cavernosa. The mitotic activity increased in cavernous tissue in the absence of dinitrosyl iron complexes. The efficiency of long-term action of dinitrosyl iron complexes on the erection in both intact animals and animals subjected to neuroectomy of cavernous tissue nerve was shown. The injection of low-molecular dinitrosyl iron complexes to the cavernous tissue resulted in the formation of protein-bound dinitrosyl iron complexes in the tissue, which were detected by the EPR technique. It is assumed that these dinitrosyl iron complexes function as a depot of nitric oxide, providing long-lasting penis erection.     

Protein synthesis in liver,skeletal muscle,and brown adipose tissue of rats fed a protein-deficient diet

Feeding protein-deficient diets to rats is known to stimulate diet-induced thermogenesis and activate brown adipose tissue (BAT). The fact that BAT protein content, unlike that of other tissues, is unnaffected by protein deficiency prompted us to measure tissue protein synthesis in vivo in animals maintained on normal- (18.8%) and low- (7.6%) protein (LP) diets. Protein synthesis was depressed in the liver of the LP rats due to a fall in RNA activity, with no change in RNA content, and synthesis was also reduced in skeletal muscle from the LP group, but this was due to decreased RNA content with no change in RNA activity. Conversely, protein synthesis, RNA, DNA, and protein content of interscapular BAT were all unaltered in protein-restricted animals. These data indicate that, unlike liver, skeletal muscle, and whole carcass, BAT protein synthesis is not reduced in protein-restricted rats, and this may be related to activation of thermogenesis in the tissue.     

Carbohydrate metabolism in sheep when adding carboxyline to their diet

The content of glycogen and glucose, as well as aldolase, phosphofructokinase, phosphoglucomutase, glucose-6-phosphatase and fructose-1,6-diphosphatase activities in liver tissue and the same activities in skeletal muscle of sheep were determined under the influence of prolonged addition of carboxyline separately and in combination with methionine, diammonium phosphate and potassium iodine to their diet. It is established that under the influence of carboxyline the glycogen content as well as aldolase and fructose-1,6-diphosphatase activities rise significantly in the liver of the tested animals. In the skeletal muscle only aldolase activity increases.     

Ca2+-activated Protease Activity in Vitamin E-Deficient Rats

The mechanism and control of protein degradation in cells are quite mysterious. We investigated the change of protease activities in animals fed a vitamin E-deficient diet. The Ca²⁺-activated protease activity was not significantly changed in vitamin E-deficient rats during the 45 weeks of the experiment. The cathepsin B activity was increased in those animals. Electron microscopic observation on the muscle of the vitamin E-deficient rats showed destruction of myofibrils at the Z-line, narrowness of myofibrils, and dispersed myofibrils. The M-line, which is known to disappear with cathepsin L treatment, was clearly observed. The phagocytosis of muscle cells by macrophages was also observed. These results show that the abnormal myofibril protein degradation in muscle tissue of vitamin E-deficient rats is not only due to the activation of macrophages and the increment of lysosomes in muscle cells, but also due to the protease which can destroy the myofibril at the Z-line. It may be a Ca²⁺-activated protease.     

Mitochondrial Ca2+ transport in lean and genetically obese (ob/ob) mice.

Isolated mitochondria from liver or brown adipose tissue of obese ob/ob mice demonstrated increased rates of Ca2+ uptake and release compared with those of lean mice. This enhanced transport activity was not found in mitochondria from kidney or skeletal muscle. Respiration-induced membrane potential was the same in mitochondria from lean and ob/ob mice. It is therefore concluded that the increased Ca2+ uptake rates reflect an activation of the Ca2+ uniporter rather than a change in the electrophoretic driving force. As mitochondria from pre-obese ob/ob mice did not show elevated rates of Ca2+ transport, the activated transport in the obese animals was thus a consequence of the state of obesity rather than being a direct effect of the ob/ob genotype. It is suggested that the enhanced activity of the Ca2+-transport pathways in liver and brown adipose tissue may alter metabolic functions in these tissues by modifying cytoplasmic or intramitochondrial Ca2+ concentrations.     

Gluconeogenesis in vertebrate livers.

1. The hypothesis is advanced that it would be logical for a tissue (liver) to evolve as a gluconeogenic organ in order to recover the lactate produced as a result of rapid and sustained contraction of skeletal muscle. 2. Lactate was present in skeletal muscle of all animals examined and increased following electrical stimulation. It was also present in the blood. 3. Gluconeogenesis from lactate occurred in liver slices of all animals excepting amphibia. However, livers of these animals also contained much glycogen and are probably gluconeogenic. 4. Phosphoenolpyruvate carboxykinase was present in all animals investigated; pyruvate carboxylase was present in all animals excepting the toad.     

The effect of fasting on the activation in vivo of the insulin receptor kinase.

Fasting causes insulin resistance in liver and fat, and increases insulin sensitivity in muscle. We studied the response in vitro and in vivo to insulin of the insulin receptor tyrosine kinase in muscle and liver from 72 h fasted and control rats. Insulin was injected intraperitoneally together with glucose, and blood and tissue samples were obtained 0, 5, 15 and 30 min later. Basal serum glucose and insulin levels were significantly higher in control than in fasting rats. Serum glucose rose to approximately 300 mg/dl at 5 min and then progressively declined without hypoglycaemia. Receptors were prepared from whole tissue by wheat germ lectin affinity chromatography. 125I-insulin binding to purified receptors was increased by fasting in both muscle (18%) and liver (50%). In untreated fasting and control animals, muscle and liver insulin receptor tyrosine kinase activity was stimulated to similar levels by insulin added in vitro. With only insulin treatment in vivo, muscle receptor tyrosine kinase behaved similarly in fasting and control animals with maximal activation at 15 min post injection. In liver, insulin in vivo stimulated receptor tyrosine kinase activity maximally at 5 min post injection in both fasting and control, but in fasting animals the treatment in vivo caused a significantly larger and more prolonged activation of the enzymic activity, possibly due to a decrease in the rate of dephosphorylation and deactivation of the beta subunits.     

AUTORADIOGRAPHIC LOCALIZATION OF NEW RNA SYNTHESIS IN HYPERTROPHYING SKELETAL MUSCLE

Work-induced growth of rat soleus muscle is accompanied by an early increase in new RNA synthesis. To determine the cell type(s) responsible for the increased RNA synthesis, we compared light autoradiographs of control and hypertrophying muscles from rats injected with tritiated uridine 12, 24, and 48 h after inducing hypertrophy. There was an increased number of silver grains over autoradiographs of hypertrophied muscle. This increase occurred over connective tissue cells; there was no increase in the number of silver grains over the muscle fibers. Quantitative studies demonstrated that between 70 and 80% of the radioactivity in the muscle that survived fixation and washing was in RNA. Pretreatment of the animals with actinomycin D reduced in parallel both the radioactivity in RNA and the number of silver grains over autoradiographs. Proliferation of the connective tissue in hypertrophying muscle was evident in light micrographs, and electron micrographs identified the proliferating cells as enlarged fibroblasts and macrophages; the connective tissue cells remained after hypertrophy was completed. Thus, proliferating connective tissue cells are the major site of the increase in new RNA synthesis during acute work-induced growth of skeletal muscle. It is suggested that in the analysis of physiological adaptations of muscle, the connective tissue cells deserve consideration as a site of significant molecular activity.     

Rapid,simple and sensitive microassay for skeletal and cardiac muscle myoglobin and hemoglobin: use in various animals indicates functional role of myohemoproteins

A novel, simple, rapid, sensitive and reproducible microassay is described for determination of myoglobin and hemoglobin content of myocardial and skeletal muscle biopsy specimens from various mammals, birds and fish. As little as 50 mg of tissue is needed and myoglobin concentrations lower than 1 mg% can be detected. Myoglobin and hemoglobin are separated at alkaline pH by ammonium sulfate extraction followed by ultrafiltration. Heme content is determined by absorption of the Soret band when the hemoprotein extract is visibly colored or more sensitively by its peroxidase activity when the extract has low color. The heme reacts with tertiary-butyl hydroperoxide and orthotolidine to generate a blue color. Hemoglobin content is correlated with myoglobin content and is related to aerobic capacity and blood flow to the tissue. Myoglobin content varied over 5 orders of magnitude up to 7 per cent of the weight of tissue, whereas hemoglobin content varied over 2 orders of magnitude up to 6 per cent of tissue weight. Myoglobin content is increased in species with high basal metabolic rate, high physical activity, prolonged diving capacity, fatigue resistance, and red muscle, whereas it is decreased in white muscle, iron-deficient animals, animals with sedentary lifestyles, and in animals and tissues with small fiber diameters such as avian or fish hearts.     

Restoration of lipoprotein lipase activity in insulin-deficient rats by insulin infusion is tissue-specific

The activity of lipoprotein lipase was measured in white and brown adipose tissues, red vastus lateralis muscle, and heart of rats that have been insulin deficient (streptozotocin, 75 mg.kg-1) for 2 weeks, and that have then received implants of insulin-delivering minipumps (17 U.kg-1.day-1) for 1 or 4 days. Normal glycemia was restored in insulin-deficient animals after 4 days of insulin treatment. Hypertriglyceridemia, but not hypercholesterolemia, was reversed after 4 days of insulin infusion. After 2 weeks of insulin deficiency, fasting lipoprotein lipase activity was lowered in all tissues studied. In white adipose tissue, lipoprotein lipase decreased to 50% of control values. After a single day of insulin infusion, even if tissue weight has not yet been greatly affected, total activity was completely restored to control levels. Enzyme activity in brown adipose tissue was also depressed in deficient animals, and insulin infusion was followed by a slow recovery of activity, to a level intermediate between those of control and insulin-deficient groups. Insulin status had milder effects on lipoprotein lipase activity in vastus lateralis muscle than in the adipose tissues. Deficient rats displayed 60% less activity than controls, and 4 days of hormone infusion only partially restored enzyme activity. There was a large loss of lipoprotein lipase in the heart following 2 weeks of insulin depletion, which was not counteracted by hormone infusion. Thus the speed and extent of recovery of lipoprotein lipase activity following hormone replacement in insulin-deficient animals varied widely among tissues. These findings suggest that insulin is part of the factors that determine the tissue specificity of lipoprotein lipase regulation.     

Histological evidences of reparative and regenerative effects of beta-adrenoceptor agonists, clenbuterol and isoproterenol, in denervated rat skeletal muscle

The aim of this study was to determine the contribution of beta-adrenoceptor activation in the reconstruction of the structural and functional organization of denervated skeletal muscle. beta-agonists, clenbuterol (1.2 mg/kg body weight) and isoproterenol (2 mg/kg body weight), administration (daily oral administration; maximum 7 days) to normal innervated rats as well as denervated animals caused muscle hypertrophy. An increase in mean fiber diameter confirmed this stimulated growth both in normal innervated and denervated rat gastrocnemius muscle. Examination of muscle nuclei from treated but normal innervated rat gastrocnemius exhibited features like large size, active nucleoplasm and an increase in their number per fiber cross section and per mm mean fiber length indicating towards an elevated biosynthetic activity in tissue in the presence of beta adrenoceptor agonists. Administration of drugs to normal innervated animals resulted in an emergence of central muscle nuclei. The hyperactive and enlarged muscle nuclei ultimately organized themselves into unusually elongated nuclear streaks. beta agonist treatment to denervated rats resulted in amelioration of atrophic state of tissue characterized by hypertrophy of muscle fibers thus lending to a restoration of structural organization of tissue. Bizarre shapes of nuclei in denervated muscle tend to recover to that characteristic to normal innervated muscle in presence of clenbuterol and isoproterenol hydrochloride. All observations were confirmed by administering butoxamine, a beta-adrenoceptor antagonist along with beta-agonists. The results suggests that both clenbuterol and isoproterenol hydrochloride are capable of mimicking normal innervation functions in skeletal muscle and thus play important role in the structural and functional reorganization of tissue. Amelioration of denervation atrophy in rat gastrocnemius in the presence of beta-agonists supports this.