Skeletal Muscle Protein and Amino Acid Metabolism in Experimental Chronic Uremia in the Rat: ACCELERATED ALANINE AND GLUTAMINE FORMATION AND RELEASE

The kinetics and factors regulating alanine and glutamine formation and release were investigated in skeletal muscle preparations from control and experimentally uremic rats. These preparations maintained phosphocreatine and ATP levels in vitro which closely approximated levels found in vivo. Alanine and glutamine release from uremic muscle were increased 45.8 and 36.0%, respectively, but tissue levels were unaltered. The increased release of alanine by uremic muscle was not accounted for by decreased rates of medium alanine reutilization via oxidation to CO₂ or incorporation into muscle protein. The maximal capacity of added amino acids such as aspartate, cysteine, leucine, and valine to stimulate net alanine and glutamine formation was the same in uremic and control muscle. Epitrochlearis preparations were partially labeled in vivo with [guanido-¹⁴C]-arginine. On incubation, preparations from uremic animals showed a 54.6% increase in the rate of loss of ¹⁴C-label in acid precipitable protein. Correspondingly, these same uremic preparations showed a 62.7% increase in ¹⁴C-label appearance in the acid-soluble fraction of muscle and in the incubation media. Insulin decreased alanine and glutamine release to an extent threefold greater in uremic than in control preparations, and increased muscle glucose uptake approximately threefold in all preparations. Although basal rates of [4,5-³H]leucine incorporation into protein were decreased 25% in uremic muscles as compared with control muscles, insulin stimulated [³H]leucine incorporation nearly equally in both preparations.     

The Impact of Streptozotocin-induced Diabetes Mellitus on Cyclic Nucleotide Regulation of Skeletal Muscle Amino Acid Metabolism in the Rat

The impact of diabetes on cyclic nucleotide-associated mechanisms regulating skeletal muscle protein and amino acid metabolism was assessed using epitrochlaris preparations from streptozotocin-induced diabetic rats. 1 nM epinephrine inhibited alanine and glutamine release from control preparations, but no inhibition was observed from diabetic preparations with <0.1 mM. 10 nM epinephrine stimulated lactate production from control muscle but stimulation in diabetic preparations was observed only at 0.1 mM. Serotonin inhibited amino acid release and stimulated lactate production equally in control and diabetic muscle. 0.1 mM epinephrine increased cyclic (c)AMP levels by 360% in control muscles, but these levels were increased only 83% in diabetic muscle. Basal-, fluoride-, and serotonin-stimulated adenylyl cyclase activities were equal in membrane preparations of diabetic and control muscle, but epinephrine-stimulated adenylyl cyclase was reduced by 60% in diabetic muscle. Carbamylcholine stimulation of alanine and glutamine release was blunted in diabetic preparations. Carbamylcholine increased cGMP levels in control but not in diabetic muscle. In diabetic muscle, guanylyl cyclase activity was 65% of control and the stimulation of cyclase activity by sodium azide was less in diabetic than control preparations. Added cGMP stimulated alanine and glutamine release from control, but not from diabetic muscle. These data suggest a loss of adrenergic and cholinergic responsiveness in diabetic muscle. Because amino acid release also showed a decreased responsiveness to added cAMP and cGMP, the presence of other derangements in the mechanism(s) of cyclic nucleotide regulation of muscle amino acid metabolism also seems likely.     

大鼠成骨细胞增殖及护骨素蛋白含量与甲状旁腺激素的调节效应

目的探讨大鼠成骨细胞增殖及护骨素蛋白含量与甲状旁腺激素的调节效应。方法选取新生清洁级健康SD大鼠作为研究对象,对其颅骨成骨细胞进行分离、培养,使用含不同浓度甲状旁腺激素的培养液进行连续培养,分别采用四甲基偶氮唑盐比色法和蛋白免疫印迹杂交法对不同时间大鼠成骨细胞增殖情况及护骨素蛋白表达情况进行测定。结果随着培养时间延长,所有组别的成骨细胞吸光度值均呈不断增加的趋势,且同一时间点的吸光度值均显著高于空白对照组,比较差异有统计学医院(P0.05)。培养液中甲状旁腺激素的浓度为10-8mol/L条件下,各时点的吸光度最高。观察各组成骨细胞中护骨素的表达情况,可见,其与甲状旁腺激素浓度无显著相关性,但空白对照组中护骨素与actin信号比显著高于其他组别,比较差异有统计学意义(P0.05)。结论甲状旁腺激素能刺激成骨细胞的增殖,并促进护骨素蛋白分泌水平下调。     

Metabolism of Parathyroid Hormone by Isolated Rat Kupffer Cells and Hepatocytes

Data from several laboratories indicate that hepatic mechanisms may have a distinctive role in the metabolism of intact hormone after secretion, a process that accounts, at least partly, for the heterogeneity of circulating parathyroid hormone. Accordingly, we studied the proteolysis of intact hormone by isolated rat Kupffer cells and hepatocytes. Kupffer cells (10⁶ cells/ml) and hepatocytes (10⁷ cells/ml) were incubated with unlabeled and ¹²⁵I-labeled bovine parathyroid hormone at 37°C for periods ranging up to 2 h. When incubated with Kupffer cells, intact hormone disappeared with a t_½ of 12±4 min. Radio-immunoassays using sequence-specific antisera showed that the dominant hormonal fragments recovered in the medium have an apparent molecular weight of ~6,000, lack amino-terminal antigenic determinants, and react in assays that specifically recognize determinants in the carboxy-terminal portion of the intact hormone. Amino-terminal fragments also were detected in high concentrations, particularly after short incubation periods. Radioiodinated fragments resulting from incubation of ¹²⁵I-labeled bovine parathyroid hormone with Kupffer cells had the same apparent size as fragments derived from the metabolism of unlabeled, intact hormone; when analyzed by Edman degradation, positions 34 and 37 of the intact hormone sequence were the amino-terminal amino acids of these dominant carboxy-terminal fragments. Hepatocytes did not hydrolyze the hormone. Thus, metabolism of parathyroid hormone by Kupffer cells results in the appearance of fragments in the media that are immunochemically indistinguishable from, and chemically identical with, those found in plasma when intact hormone is injected intravenously. This indicates that the proteolysis observed in vitro accurately reflects the metabolism of the hormone in vivo. The detection of amino-terminal fragments in concentrations nearly equal to those of carboxy-terminal fragments indicates that cleavage of intact hormone is, initially, by an endopeptidase(s).     

Effects of Hepatectomy, Nephrectomy, and Nephrectomy/Uremia on the Metabolism of Parathyroid Hormone in the Rat

Reports from several laboratories, showing extensive hepatic extraction of circulating parathyroid hormone, led us to examine the effect of near-total hepatectomy on the metabolism of the hormone to circulating fragments, and on its clearance from plasma. The rate of disappearance of ¹²⁵I-labeled and unlabeled bovine parathyroid hormone from plasma, and the appearance, disappearance, and chemical and immunochemical characteristics of circulating fragments were examined by gel filtration and either sequence-specific radioimmunoassays or sequence analysis using the Edman reaction. Results from awake rats subjected to near-total hepatectomy were compared with those found in sham-treated, nephrectomized, and short-term uremic rats (studied 2 d after nephrectomy). When compared with the sham-treated group, all other groups clear ¹²⁵I-labeled hormone more slowly; after hepatectomy, however, the clearance rate is most strikingly decreased.     

Branched Chain Amino Acid Oxidation in Cultured Rat Skeletal Muscle Cells: SELECTIVE INHIBITION BY CLOFIBRIC ACID

Leucine metabolism in skeletal muscle is linked to protein turnover. Since clofibrate is known both to cause myopathy and to decrease muscle protein content, the present investigations were designed to examine the effects of acute clofibrate treatment on leucine oxidation. Rat skeletal muscle cells in tissue culture were used in these studies because cultivated skeletal muscle cells, like muscle in vivo, have been shown to actively utilize branched chain amino acids and to produce alanine. The conversion of [1-¹⁴C]leucine to ¹⁴CO₂ or to the [1-¹⁴C]keto-acid of leucine (α-keto-isocaproate) was linear for at least 2 h of incubation; the production of ¹⁴CO₂ from [1-¹⁴C]leucine was saturable with a K_m = 6.3 mM and a maximum oxidation rate (V_max) = 31 nmol/mg protein per 120 min. Clofibric acid selectively inhibited the oxidation of [1-¹⁴C]leucine (K_i = 0.85 mM) and [U-¹⁴C]isoleucine, but had no effect on the oxidation of [U-¹⁴C]glutamate, -alanine, -lactate, or -palmitate. The inhibition of [1-¹⁴C]leucine oxidation by clofibrate was also observed in the rat quarter-diaphragm preparation. Clofibrate primarily inhibited the production of ¹⁴CO₂ and had relatively little effect on the production of [1-¹⁴C]keto-acid of leucine. A physiological concentration—3.0 g/100 ml—of albumin, which actively binds clofibric acid, inhibited but did not abolish the effects of a 2-mM concentration of clofibric acid on leucine oxidation. Clofibrate treatment stimulated the net consumption of pyruvate, and inhibited the net production of alanine. The drug also increased the cytosolic NADH/NAD⁺ ratio as reflected by an increase in the lactate/pyruvate ratio, in association with a decrease in cell aspartate levels. The changes in pyruvate metabolism and cell redox state induced by the drug were delayed compared with the nearly immediate inhibition of leucine oxidation. These studies suggest that clofibric acid, in concentrations that approximate high therapeutic levels of the drug, selectively inhibits branched chain amino acid oxidation, possibly at the level of the branched chain keto-acid dehydrogenase.     

Effects of Reduced Renal Mass and Dietary Protein Intake on Amino Acid Release and Glucose Uptake by Rat Muscle In Vitro

Epitrochlearis muscles obtained from normal male Holtzman rats used as controls (C) and rats with reduced renal mass (Nx) fed isocaloric diets of varying protein content were incubated in Krebs-Ringer buffer containing 5 mM glucose for 1 or 3 h with or without insulin.Alanine (ALA) release rates from muscles of Nx rats were increased 40% above C values after 1 h of incubation regardless of protein intake. Addition of insulin decreased the ALA release from muscles of Nx rats to C values in animals fed 10 and 20% casein and chow but did not in rats fed 40% casein. After 3 h of incubation, all ALA release rates decreased by congruent with40%. The ALA release from muscles of Nx rats fed 10% casein was comparable to C values and decreased further with the addition of insulin. On the other hand, ALA release from muscles of Nx rats fed 20 and 40% casein as well as chow remained significantly elevated above C values, but responded to the addition of insulin with a reduction in release rates to C values, except from the muscles of Nx animals fed 40% casein.Tyrosine (TYR) and phenylalanine (PHE) release rates also were increased in muscles from Nx rats compared with C after 1 h of incubation. Release rates were highest in the Nx group fed 10% casein and decreased with increasing protein intake. Addition of insulin decreased the release rates of Nx rats to C values in each group. After 3 h of incubation, release rates of TYR and PHE in muscles from Nx rats remained significantly above C values for all groups, but responded to the addition of insulin with a decrease to C values. Glutamine and glutamate release were not significantly affected by reduction in renal mass.Base-line glucose uptake by all groups of muscles from Nx rats was significantly greater than corresponding C values, but maximal insulin-stimulated glucose uptake was comparable in all groups. Tissue pool sizes for glycogen, ATP, phosphocreatine, ALA, glutamate, and glutamine were unaffected by reduction in renal mass.The results indicate that Nx is associated with accelerated ALA, TYR, and PHE release from muscle. ALA release rose with increasing protein intake and decreased to values observed from C muscles after addition of insulin except in Nx animals fed 40% casein. TYR and PHE release decreased with increasing protein intake and also decreased to C values with the addition of insulin. The data also suggest that ALA release is not dependent upon glucose uptake in muscles from either C or Nx rats.     

运动对骨骼肌代谢的影响

受运动强度、运动量和运动状况等因素的影响,骨骼肌基本代谢发生变化,出现结构和功能上的适应表现在骨骼肌细胞内的收缩成分、肌糖原、肌红蛋白增多,线粒体体积、数量增加,以及毛细血管分布在骨骼肌上的密度加大等,使骨骼肌的有氧代谢能力加强,肌肉收缩效率提高,运动到力竭的时间延长。同时指出急性运动时肌浆网结构改变和功能下降是诱发骨骼肌疲劳的重要原因,而高强度训练却可以提高肌浆网功能及Ca     

Enzymatic and Metabolic Studies on Carbohydrate and Amino Acid Metabolism in Rat Liver during Acute Uraemia

Abstract. Enzyme activities of the glycolytic, gluconeogenic, and hexose monophosphate pathways were measured in the liver of starved rats 12 and 48 hours after bilateral nephrectomy. Control experiments (sham operated rats) revealed that alterations of enzyme activities were not due to uraemia but to starvation. Alanine-aminotransferase and aspartate- aminotransferase activities, however, were significantly elevated in rat liver 48 hours after nephrectomy when compared with sham operated controls. Concentrations of some of the gluconeogenic intermediates (3-phosphoglyceric acid, pyruvate, phosphoenolpyruvate and glucose-6-phosphate) were significantly higher in the liver of uraemic animals. Amino acid analysis showed an increase in only L-alanine concentration. It is suggested that the elevated content of pyruvate in the liver during acute uraemia is due to an inhibition of pyruvate degradation. Together with the elevated pyruvate concentration the increase in L-alanine could be explained as a consequence of the equilibrium of the alanine-aminotransferase reaction; K_app. of the reaction is not changed by uraemia. Increased activities of the transaminases and the elevated concentrations of the other metabolites measured might indicate that in the liver of nephrectomized rats there is enhanced gluconeogenesis from substrates other than pyruvate.     

Biosynthesis of Proparathyroid Hormone and Parathyroid Hormone by Human Parathyroid Glands

Human parathyroid glands obtained at autopsy were incubated with [(3)H]leucine and [(3)H]lysine. After incubation, nonradioactive parathyroid tissue of either human or bovine origin was added. Radioactive parathyroid hormone and proparathyroid hormone were isolated from the gland and medium by organic solvent and salt fractionation, trichloroacetic acid precipitation, Sephadex G-100 gel filtration, and carboxymethyl cellulose column chromatography. The human hormonal peptides were identified in the ion-exchange column eluates by their relatively high levels of radioactivity, their elution positions, and their immunoreactivity to anti-PTH antiserum. The time-course of radioactive amino acid incorporation into these peptides and a brief incubation of the gland with radioactive amino acids, followed by various lengths of incubation with nonradioactive amino acids, indicated that a precursor-product relationship exists for the two peptides. An alternate method for isolation of the hormone and prohormone, which involves separation of peptides by urea-polyacrylamide gel electrophoresis, confirmed the identities of the human parathyroid hormone and proparathyroid hormone.     

Effect of Thyroid Status on Insulin Action in Rat Adipocytes and Skeletal Muscle

Isolated adipocytes and soleus muscles prepared from mature rats, rendered hypothyroid by a low iodine diet and propylthiouracil, markedly resisted the ability of insulin to increase glucose utilization. In adipocytes, the sum of basal d-(1-(14)C)-glucose conversion to CO(2), glyceride-glycerol, and fatty acid was unaltered by hypothyroidism, although conversion to fatty acid was decreased. The response of each of these metabolic pathways to insulin at all concentrations tested was greatly diminished in hypothyroid rat adipocytes. 3-O-Methylglucose transport rates in the presence of insulin were not significantly different in adipocytes from hypothyroid as compared with euthyroid rats, although basal transport rates were significantly higher in the hypothyroid state. Lipolysis and cyclic AMP accumulation in adipocytes from hypothyroid rats in response to theophylline were markedly diminished compared with euthyroid controls, but insulin was about as effective in inhibiting lipolysis in these cells as in those derived from euthyroid animals. The binding of (125)I-insulin to adipocytes at several hormone concentrations was also shown to be unaffected by hypothyroidism.In soleus muscle, basal glucose conversion to H(2)O and glycogen was unaltered in the hypothyroid state, whereas insulin action on these pathways was markedly inhibited. The decrease in muscle insulin responsiveness was less marked than that observed in adipocytes. Uptake of either 2-deoxyglucose or l-arabinose in the presence or absence of insulin was similar in soleus muscles derived from euthryoid vs. hypothyroid rats. Similarly, insulin action on the conversion of soleus muscle glycogen synthase D to the I form in the absence of glucose was unaltered by hypothyroidism. We conclude that (a) hypothyroidism in mature rats leads to a marked decrease in the responsiveness of glucose metabolism in adipocytes and skeletal muscle to insulin; (b) no detectable impairment of the membrane insulin effector systems that mediate the regulation of adipocyte hexose transport and glycogen synthase is caused by hypothyroidism in this animal model; and (c) the cellular defect that leads to apparent insulin resistance of adipocyte and soleus muscle glucose utilization resides at the level of one or more intracellular enzymes involved in glucose catabolism.     

Nalidixic Acid and Macromolecular Metabolism in Tetrahymena pyriformis: Effects on Protein Synthesis

A study on the effect of nalidixic acid on macromolecular metabolism, particularly of protein, in Tetrahymena pyriformis was performed. It was shown that the compound is a potent inhibitor of deoxyribonucleic acid, ribonucleic acid, and protein synthesis for this organism. A conspicuous breakdown of polysomes, accompanied by the accumulation of 80S ribosomes, occurred in cells incubated for 10 min with the drug; polysome formation was prevented. The accumulating 80S particles were shown to be run-off ribosomal units. The incorporation of amino acids by a cell-free system is not affected by nalidixic acid. In nonproliferating cells the incorporation was also not prevented, unless the cells were previously incubated with the drug. These results are discussed in terms of the possible mechanism of action of nalidixic acid in T. pyriformis.     

Skeletal Muscle Calcium Metabolism and Contractile Force in Vitamin D-deficient Chicks

The myopathy associated with vitamin D deficiency has not been well characterized, and it is not known if weakness is a result of a specific effect of vitamin D deficiency on skeletal muscle. Chicks were raised from hatching on a vitamin D-deficient diet, and by 3 wk of age were hypocalcemic and appeared weak. Tension generated by triceps surae during repetitive stimulation of posterior tibial nerve was significantly less than that developed by chicks given vitamin D(3) supplements (309 g tension/g wet weight of triceps surae, SD 60, for vitamin D-deficient chicks; 470, SD 77, for vitamin D(3)-treated chicks, P < 0.01). Histochemical and electron microscopic examination of skeletal muscles of these chicks showed no abnormalities, and there were no electrophysiologic evidences of motor nerve or neuromuscular junction dysfunction. The concentration of ATP in skeletal muscle of the vitamin D-deficient chicks (5.75 mumol/g wet weight, SD 0.17) was not significantly different from that in vitamin D-treated chicks (5.60, SD 0.50). There was no correlation between strength and serum calcium, serum inorganic phosphate, or skeletal muscle inorganic phosphate. Relaxation of tension after tetanic stimulation was slowed in the vitamin D-deficient chicks (20.6 ms, SD 1.7, vs. 15.4, SD 1.3, in vitamin D-treated chicks and 15.3, SD 1.0, in normal control chicks), and in vitro (45)Ca(++) transport by sarcoplasmic reticulum from the vitamin D-deficient chicks was reduced. Calcium content of mitochondria prepared from leg muscles of vitamin D-deficient chicks (24 nmol/mg mitochondrial protein, SD 6) was considerably lower than that of mitochondria from normal control chicks (45, SD 8) or from chicks treated with vitamin D for 2 wk or more (66-100, depending upon level and duration of therapy). Treatment of the vitamin D-deficient chicks from hatching with sufficient dietary calcium to produce hypercalcemia did not significantly raise skeletal muscle mitochondrial calcium content (31 nmol/mg mitochondrial protein, SD 7) and did not prevent weakness. These studies demonstrate objective weakness as a result of myopathy in vitamin D-deficient chicks, and provide evidence that vitamin D deficiency has effects on skeletal muscle calcium metabolism not secondary to altered plasma concentrations of calcium and phosphate.     

Metabolic Activity in Rat Skeletal Muscle:Effect of Intermittent Hypoxia

Abstract. In order to determine whether hypoxia is a trigger for changed metabolic activity in muscle tissue, rats were exposed to intermittent hypoxia of varying degree (5% 0₂, 8% 0₂, 10% 0₂, 12% 0₂, 15% 0₂ in N₂) 3 h per day for 1–4 weeks. The effects of hypoxia on the rate of incorporation of glucose-carbon into glycogen, lipids, carbon dioxide and lactate and on succinic oxidase activity in skeletal muscle tissue were determined. Rats intermittently exposed to 5% oxygen in nitrogen for one week showed a significant decrease of the rate of incorporation of glucose-carbon into lipids and carbon dioxide and a significant decrease in succinic oxidase activity. In rats exposed to 8 and 10% 0₂ a significant increase of the incorporation rate of glucose-carbon into glycogen, lipids and carbon dioxide was found after 1 and 4 weeks and succinic oxidase activity increased after 4 weeks. The phospholipid concentration in muscle tissue decreased after 8 and 10% 0₂ exposure for 4 weeks. In the rats exposed to 8% oxygen for 4 weeks the protein concentration of muscle tissue increased significantly.–Based on these results it is suggested that the change of the glycolytic activity and succinic oxidase activity in muscle tissue after physical training and in arterial insufficiency are induced by hypoxia in the muscles.     

老年大鼠骨骼肌肌源细胞的纯化及培养

【目的】探讨老年大鼠骨骼肌肌源细胞纯化及培养方法。【方法】取老年雌性SD大鼠前肢肱三头肌,用胶原酶和Dispase进行消化,采用差速贴壁的方法纯化骨骼肌肌源细胞,用含20%胎牛血清的HamsF10培养基进行培养,α-骨骼肌肌动蛋白（-αsarcometric actin）免疫细胞化学染色进行细胞鉴定。【结果】成功地分离培养了老年大白鼠的骨骼肌肌源细胞。【结论】采用差速贴壁的方法可以成功纯化骨骼肌肌源细胞,为肌源细胞自体移植治疗压力性尿失禁打下了基础。     

Concentration of Myo-inositol in Skeletal Muscle of the Rat Occurs without Active Transport

The cellular uptake of nonphosphorylated myo-inositol (MI) and its incorporation into phosphoinositide in the rat epitrochlearis muscle was measured. Cellular uptake of [2-³H]MI was determined by the difference between total uptake and [2-³H]MI present in the extracellular fluid determined with [1-¹⁴C]mannitol. Cellular uptake was parabolic and directly proportional to medium MI concentrations between 25 and 3,200 μM. Saturation of a MI carrier was not evident. Moreover, uptake was not inhibited by 2 mM ouabain, 0.3 mM 2,4-dinitrophenol, or 22 mM glucose. Insulin, 100 mU/ml, was without effect on either cellular uptake of [2-³H]MI or its incorporation into phosphoinositides. In muscles that were preloaded with [2-³H]MI and then incubated in media that contained a constant amount of MI but no [2-³H]MI, 44.3% of the [2-³H]MI was released after 10 min increasing to 62.5% by 120 min. Cellular MI concentrations were 0.18 μmol/g wet tissue (four times plasma levels) in rapidly isolated and frozen epitrochlearis muscle. When muscle was incubated without MI, 48% of endogenous MI was lost rapidly. Restoration of cellular MI in 50 μM MI media occurred in two phases, a rapid uptake phase lasting 10 min and a subsequent slow phase of MI uptake.     

Inhibitory Effect of Epinephrine on Insulin-stimulated Glucose Uptake by Rat Skeletal Muscle

The effect of epinephrine on basal and insulin-stimulated glucose uptake in perfused hindlimbs of fed rats was studied. Insulin increased glucose uptake in a dose-dependent manner from a basal value of 1.5±0.3 up to a maximum value of 5.3±0.9 μmol/min per 100 g with 6 nM (1 m U/ml). Epinephrine at 10 nM and 0.1 μM also increased glucose uptake to 2.6±0.1 and 3.1±0.1 μmol/min per 100 g, respectively. These same concentrations of epinephrine, however, suppressed the insulin-stimulated glucose uptake to 3.2±0.3 μmol/min per 100 g. Both the stimulatory and inhibitory effects of epinephrine on glucose uptake were completely reversed by propranolol, but were not significantly altered by phentolamine.