The effects of amino acids on albumin synthesis by the isolated perfused rat liver

Albumin synthesis was measured in the isolated perfused rat liver by using the livers of both well-fed and starved rats. Starvation markedly decreased albumin synthesis. The livers from starved rats were unable to increase synthesis rates after the addition to the perfusates of single amino acids or the addition of both glucagon and tryptophan. Arginine, asparagine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, threonine, tryptophan and valine, added together to ten times their normal peripheral blood concentrations, restored synthesis rates to normal. The plasma aminogram (i.e. the relative concentrations, of amino acids) was altered by depriving rats of protein for 48h. The use of blood from the deprived rats as perfusate, instead of normal blood, decreased albumin synthesis rates significantly by livers obtained from well-fed rats. The addition of single amino acids, including the non-metabolizable amino acid, alpha-aminoisobutyric acid, to the above mixture increased albumin synthesis rates to normal values. It is concluded that amino acids play an important role in the control of albumin synthesis and that more than one mechanism is probably involved.     

Contrasting response of protein degradation to starvation and insulin as measured by release of N tau-methylhistidine or phenylalanine from the perfused rat heart.

An isotope-dilution method is described for the measurement of N tau-methylhistidine release from the perfused rat heart. We argue that release of N tau-methylhistidine is indicative of cardiac actin degradation. N tau-Methylhistidine release is compared with phenylalanine release in the presence of cycloheximide (phenylalanine release being a measure of degradation of mixed proteins). In hearts perfused with glucose plus acetate, the rate of actin degradation was increased by starvation and was not inhibited by insulin. In contrast, the rate of mixed-protein degradation was decreased by starvation and was inhibited by insulin. The fractional rate of degradation of mixed proteins in hearts from fed or starved rats was greater than that for actin. It is suggested that there are at least two pools of intracellular protein, the degradation rates of which differ in terms of their response to insulin and starvation.     

Protein turnover in pulmonary macrophages. Utilization of amino acids derived from protein degradation.

Conditions were defined under which rates of protein synthesis and degradation could be estimated in alveolar macrophages isolated from rabbits by pulmonary lavage and incubated in the presence of plasma concentrations of amino acids and 5.6 mM-glucose. Phenylalanine was validated as suitable precursor for use in these studies: it was not metabolized appreciably, except in the pathways of protein synthesis and degradation; it entered the cells rapidly; it maintained a stable intracellular concentration; and it was incorporated into protein at measurable rates. When extracellular phenylalanine was raised to a concentration sufficient to minimize dilution of the specific radioactivity of the precursor for protein synthesis with amino acid derived from protein degradation, the specific radioactivity of phenylalanyl-tRNA was only 60% of that of the extracellular amino acid. This relationship was unchanged in cells where proteolysis increased 2.5-fold after uptake and degradation of exogenous bovine serum albumin. In contrast, albumin prevented the decrease in phenylalanine incorporation observed in macrophages deprived of an exogenous source of amino acids. These observations suggested that macrophages preferentially re-utilized amino acids derived from the degradation of endogenous, but not from exogenous (albumin), protein. However, when the extracellular supply of amino acids was restricted, substrates derived from albumin catabolism could support the protein-synthetic pathway.     

Albumin catabolism in diabetic rats

The kinetics of albumin catabolism were studied in normal rats and rats with streptozotocin induced diabetes (blood glucose greater than 500 mg%). Whether determined from the clearance of 125I-albumin from plasma or from the whole body, after 10 days of severe, uncontrolled diabetes there was a 30-35% decrease in the catabolic rate for albumin in the diabetic rats compared to normals. There was also about a 35% contraction of the relative extravascular distribution volume for albumin in the diabetic rats, and about a 25% decrease in the total body mass of albumin. However, the concentration of albumin in the circulation was the same in normal and diabetic animals. We conclude that when the rate of albumin synthesis is substantially depressed in diabetes, the rat maintains normal plasma albumin concentration both by decreasing albumin's fractional catabolic rate and by shifting albumin from the extravascular to the vascular compartment.     

Biliary protein output by isolated perfused rat livers. Effects of bile salts.

The output of proteins into bile was studied by using isolated perfused rat livers. Replacement of rat blood with defined perfusion media deprived the liver of rat serum proteins (albumin, immunoglobulin A) and resulted in a rapid decline in the amounts of these proteins in bile. When bovine serum albumin was incorporated into the perfusion medium it appeared in bile within 20 min and the amount in the bile was determined by the concentration of the protein in the perfusion medium. The use of a defined perfusion medium also deprived the livers of bile salts and the amounts of these, and of plasma-membrane enzymes [5'-nucleotidase (EC 3.1.3.5) and phosphodiesterase I], in bile declined rapidly. Introduction of micelle-forming bile salts (taurocholate or glycodeoxycholate) to the perfusion medium 80 min after liver isolation markedly increased the output of plasma-membrane enzymes but had no effect on the other proteins. The magnitude of this response was dependent on the bile salt used and its concentration in bile; there was little effect on plasma-membrane enzyme output until the critical micellar concentration of the bile salt had been exceeded in the bile. A bile salt analogue, taurodehydrocholate, which does not form micelles, did not produce the enhanced output of plasma-membrane enzymes. This work supports the view that the output of plasma-membrane enzymes in bile is a consequence of bile salt output and also provides evidence for mechanisms by which serum proteins enter the bile.     

Protein turnover,synthesis and secretion of albumin in hepatocytes isolated from rats bearing walker 256 carcinoma

Summary Hepatocytes isolated from rats bearing line A of Walker 256 carcinoma (WA) were used to study the turnover of total liver protein and the synthesis of albumin in comparison with ad libitum (AL) and pair-fed (PF) healthy controls. The rates of total protein synthesis by hepatocytes of WA animals were 40 and 90% higher than in AL and PF controls, respectively. The degradation of fast-turnover proteins was not affected by nutrition or by the tumor, whereas the degradation of slow-turnover proteins was slightly but significantly increased—about 24% higher in hepatocytes from WA rats than in PF controls. The combination of the two processes, synthesis and degradation, was in favor of an increased synthesis which explains the increase in liver protein content observed in vivo in WA rats. Dietary restriction did not affect the synthesis and secretion of albumin, whereas the tumor significantly reduced its synthesis by about 30%. The plasma concentration of albumin in WA rats dropped by about the same percentage compared with AL and PF animals.     

Binding and transport of transthyretin-gold by the endothelium of the rat choriocapillaris

The photoreceptors of the neural retina require retinol for synthesis of rhodopsin. In the plasma, retinol is bound to retinol binding protein which is carried by transthyretin (TTR; formerly called prealbumin). It is unknown whether, or how, retinol carrier proteins cross the endothelium of the choriocapillaris, the blood supply to the outer neural retina. This was examined in the present study with TTR-gold probes perfused into rats and localized by electron microscopic techniques. TTR-gold, often in clusters, was localized to diaphragmed fenestrae, parajunctional areas, coated pits, transendothelial channels, multivesicular bodies, and to vesicles close to the Golgi apparatus. The probe was also identified at the luminal and abluminal fronts and the interior of transendothelial channels in an apparent sequence of transit. TTR-gold was also found in a series of interconnected vesicles adjacent to the abluminal side of the endothelium. Localizations were not seen when rat albumin fraction V was substituted for TTR and when the rats were perfused with Pronase E before labeling with TTR-gold. These observations indicate that binding and receptor mediated-like transport of TTR by the endothelium of the choriocapillaris is present. This is similar to the processing of heparin-gold by this endothelium.     

Rates of protein turnover in vivo and in vitro in ventricular muscle of hearts from fed and starved rats.

Starvation of 300 g rats for 3 days decreased ventricular-muscle total protein content and total RNA content by 15 and 22% respectively. Loss of body weight was about 15%. In glucose-perfused working rat hearts in vitro, 3 days of starvation inhibited rates of protein synthesis in ventricles by about 40-50% compared with fed controls. Although the RNA/protein ratio was decreased by about 10%, the major effect of starvation was to decrease the efficiency of protein synthesis (rate of protein synthesis relative to RNA). Insulin stimulated protein synthesis in ventricles of perfused hearts from fed rats by increasing the efficiency of protein synthesis. In vivo, protein-synthesis rates and efficiencies in ventricles from 3-day-starved rats were decreased by about 40% compared with fed controls. Protein-synthesis rates and efficiencies in ventricles from fed rats in vivo were similar to values in vitro when insulin was present in perfusates. In vivo, starvation increased the rate of protein degradation, but decreased it in the glucose-perfused heart in vitro. This contradiction can be rationalized when the effects of insulin are considered. Rates of protein degradation are similar in hearts of fed animals in vivo and in glucose/insulin-perfused hearts. Degradation rates are similar in hearts of starved animals in vivo and in hearts perfused with glucose alone. We conclude that the rates of protein turnover in the anterogradely perfused rat heart in vitro closely approximate to the rates in vivo in absolute terms, and that the effects of starvation in vivo are mirrored in vitro.     

The hypoalbuminaemia of ovine fascioliasis: the influence of protein intake on the albumin metabolism of infected and of pair-fed control sheep.

Studies using ¹²⁵I-albumin and ⁵¹Cr-labelled plasma proteins showed that the hypoalbuminaemia which developed in sheep during the migratory stage of Fasciola hepatica infections was brought about by a combination of reduced albumin synthesis and plasma volume expansion. It was suggested that these changes were a reflection of the attendant liver damage and possibly of preferential utilisation of amino acids for immunoglobulin production. During the biliary stage of the disease, when the animals developed even more marked hypoalbuminaemia, increased albumin degradation arising from excessive plasma leakage into the gut were the outstanding features. The severity of these changes was closely linked to the state of the albumin pools which in turn was related to such factors as the plane of nutrition, appetite and fluke burden of the host. More albumin was catabolised by sheep with low fluke burdens, and in animals with the same level of infection, greater rates of catabolism were associated with a high protein intake. Sheep which catabolised most albumin became the least hypoalbuminaemic and survived longest. These animals also synthesised most albumin. It was shown that by impairing albumin synthesis, inappetence was an important additional factor in the hypoalbuminaemia of heavy infections, particularly if superimposed on a low protein diet. Nevertheless, irrespective of the size of their adult fluke burden, chronically-infected sheep were able to synthesise more albumin than pair-fed controls.     

Reversible inhibition of protein synthesis in lung by halothane

Alterations in the synthesis and degradation of proteins were investigated in intact lungs exposed to the volatile anaesthetic halothane. In rat lungs perfused in situ with Krebs-Henseleit bicarbonate buffer containing 4.5% (w/v) bovine serum albumin, 5.6 mM-glucose, plasma concentrations of 19 amino acids and 690 microM-[U-14C]-phenylalanine and equilibrated with O2/N2/CO2 (4:15:1), protein synthesis, calculated based on the specific radioactivity of aminoacyl-tRNA, was inhibited by halothane. The anaesthetic did not affect degradation of lung proteins. The inhibition of protein synthesis was rapid in onset, dose-dependent, and quickly reversible. It did not appear to be associated with overall energy depletion, with non-specific changes in cellular permeability, or with decreased availability of amino acids as substrates for protein synthesis.     

Quantification of the hepatic contribution to the catabolism of high density lipoproteins in rats.

Isolated rat livers were perfused for four hours in a recirculating system containing washed rat erythrocytes. Biologically screened radioiodinated rat high density lipoproteins (1.090 < d < 1.21 g/ml) were added to the perfusate with different amounts of whole serum to supply unlabeled rat high density lipoproteins. The protein moiety of the lipoprotein contained more than 95% of the radioiodine. The fraction of apolipoprotein mass degraded during the perfusion was quantified by the linear increment of non-protein-bound radioiodine in the perfusate, corrected for the increment observed during recirculation of the perfusate in the absence of a liver. The small amount of (131)I secreted into bile was added to calculate the fractional catabolic rate. The fractional catabolic rate ranged from 0.22 to 0.63% per hour in 12 experiments and was inversely related to the size of the perfusate pool of high density apolipoprotein. The absolute catabolic rate of high density apolipoprotein (fractional catabolic rate x pool size) in three livers in which the concentration of rat HDL in the perfusate approximated that in intact rats was 69.5 +/- 10.4 micro g hr(-1) (mean +/- SD). The rate of disappearance of cholesteryl esters of rat high density lipoproteins (labeled biologically by injecting donor rats with [5-(3)H]mevalonic acid) from the liver perfusate did not exceed that of the apoprotein component. These rates were compared with catabolic rates for rat high density lipoproteins in intact rats. Fractional catabolic rate in vivo, obtained by multicompartmental analysis of the disappearance curve of (131)I-high density apolipoprotein from blood plasma, was 11.9 +/- 1.3% hr(-1) (mean +/- SD). Total catabolic rate in vivo (fractional catabolic rate x intravascular pool of high density apolipoprotein) was 986 +/- 145 micro g hr(-1) (mean +/- SD). The results suggest that only a small fraction of high density lipoproteins in blood plasma of rats is degraded directly by the liver.-Sigurdsson, G., S-P. Noel, and R. J. Havel. Quantification of the hepatic contribution to the catabolism of high density lipoproteins in rats.     

Quantitative Significance of Plasma Albumin Metabolism in the Whole Body Protein Turnover in Rats

The amount of extra- and intravascular albumin was estimated in two groups of rats, i.e., those fed a 20% casein (20% protein) diet and a 3% casein (low protein or 3% protein) diet.

The fractional turnover rate of whole body plasma albumin was also measured in the two groups of rats, employing the constant infusion method of Waterlow et al. At the same time, the fractional turnover rate of the whole body protein was measured.

When the diet was changed from the 20% protein to the 3% protein diet, the amount of albumin mass in both extra- and intravascular spaces decreased significantly. During 7 days on the diet, the extra- and intravascular albumin mass per 100 g of body weight did not change significantly in the rats fed the 20% protein diet. On the other hand, rats fed the 3% protein diet lost almost 30% of the extra- and intravascular albumin per lOOg body weight.

The fractional turnover rates of whole body albumin were estimated to be 31.7 and 19.8%/day in the 20% protein and the 3% protein diet-fed rats, respectively. The fractional turnover rates of whole body protein were 16.1 and 10.6%/day in the 20% protein and the 3% protein diet-fed rats, respectively.

The leucine fluxes to albumin synthesis and whole body protein synthesis were calculated to be 5.9 and 83 μmol/hr, respectively, in the 20% protein diet-fed rats. The leucine fluxes in the 3% protein diet-fed rats were 2.5 and 54μmol/hr for the albumin synthesis and for the whole body protein synthesis, respectively.

These results demonstrate the quantitative significance of albumin metabolism in the whole body protein turnover in rats fed on two levels of protein intake.     

Role of changes in protein degradation in the growth of regenerating livers.

The significance of changes in rates of synthesis, export, and degradation of proteins during liver regeneration was assessed. (a) Proteins were pulse labeled by the intravenous injection of radioactive leucine and, 5 min later, pactamycin (an inhibitor of the initiation of protein synthesis). One-half of the protein radioactivity was lost from the normal liver within 3 hours. From the radioactivity of the plasma proteins at that time and a study of the disappearance of these proteins from the circulation, it was calculated that 28% of the newly synthesized proteins were exported. Serum albumin accounted for a third of the exported proteins. Thirty-six hours after partial hepatectomy the proportion of albumin to total protein synthesis remained constant, while that of the other plasma proteins increased by 50%. The fraction of the newly synthesized proteins retained by the liver after 3 hours decreased by 20%. (b) During the first 36 hours of liver regeneration the average rates of protein degradation slowed down to one-half the normal values. This was determined either by the loss of radioactivity from total protein (or the guanidino-C of protein-bound arginine) in livers labeled with [14C]bicarbonate, or calculated as the balance between protein synthesis and net protein gain. (c) From these results, and those of our previous study of the protein synthetic machinery of normal and regenerating livers (Scornik, O.A. (1974)J. Biol. Chem. 249, 3876-3883), we conclude that changes in the rate of protein degradation are the single most important factor determining the increase in protein content during liver compensatory growth.     

Phe(13),Tyr(19)-melanin-concentration hormone and the blood-brain barrier: role of protein binding

Melanin-concentrating hormone (MCH), found both peripherally and centrally, is involved in food ingestion. Although its expression in brain is increased by fasting, it is not known whether it crosses the blood-brain barrier (BBB). Use of the sensitive method of multiple-time regression analysis has shown that almost all of the peptides and polypeptides tested cross the BBB at a rate faster than the vascular marker albumin. With this same method, however, we found that the 19-amino acid 125I-Phe13,Tyr19-MCH did not cross faster than 99mTc-albumin. Several mechanisms were excluded as possible explanations for the slow rate of influx. These included degradation, association with capillary endothelial cells, and transport from brain to blood. When Phe13,Tyr19-MCH was perfused in blood-free buffer, however, it entered the brain significantly faster than albumin. This suggested protein binding as an explanation for the slow rate of influx when the MCH was administered in blood. Protein binding was confirmed by capillary zone electrophoresis, which showed that almost all of the Phe13,Tyr19-MCH added to blood migrated with a large-molecular-weight substance. Sodium dodecyl sulfate-capillary gel electrophoresis of Phe13,Tyr19-MCH in buffer additionally showed that the MCH aggregated as a trimer, a factor not preventing its influx by blood-free perfusion. Thus, the results show that blood-borne Phe13,Tyr19-MCH does not significantly cross the BBB, probably because of its binding to serum proteins.     

Regulation of protein secretion by crinophagy in perfused rat liver.

We examined the secretion of three serum proteins, albumin (RSA), alpha 2 mu-globulin (alpha 2 mu G), and transferrin (Trf), in the isolated perfused liver. Within 4 h of perfusion, only 20 to 35% of previously synthesized proteins were secreted by the liver into the recirculating medium. Low temperature inhibited the secretion of alpha 2 mu G and Trf, but not RSA. The amount of RSA secreted by the liver increased twofold in the presence of leupeptin, a proteinase inhibitor, or primaquine, a weak base capable of neutralizing acidic compartments. Neither drug affected Trf secretion, while the release of alpha 2 mu G was enhanced threefold by primaquine treatment. Only 55 to 70% of the total amount of these serum proteins present in the liver at the onset of perfusion could be accounted for after 4 h of perfusion. Our evidence suggests that these losses are due to protein degradation. The degradation of RSA and alpha 2 mu G was inhibited at 15 degrees C and by both leupeptin and primaquine. Contrary, RSA degradation was not altered when livers were perfused at 20 degrees C. Morphological techniques combined with immunological probes were utilized to identify possible intracellular sites of RSA degradation. RSA and cathepsin L were colocalized to large vacuoles found near the cell periphery. Entry of RSA into these vacuoles occurred at 20 degrees C but not at 15 degrees C. Our results using perfused rat livers suggest that as much as 40% of hepatic serum proteins are degraded via fusion of secretory vesicles with lysosomes (e.g., crinophagy).     

Identification of albumin as the plasma carrier for zinc absorption by perfused rat intestine.

The isolated vascularly perfused rat intestine exhibits an obligatory need for a protein carrier in order to absorb zinc. Therefore this system is ideal for use as a model to identify the plasma carrier during zinc absorption. Affinity chromatography on Blue Sepharose CL-6B was employed to separate the major serum zinc-binding proteins in the portal effluent of the perfused intestine. It was found that 94% of newly absorbed 65Zn was transported in the portal serum-containing perfusate as an albumin-65Zn complex. The identity of albumin as the plasma carrier was confirmed by polyacrylamide-slab-gel electrophoresis. This evidence suggests that albumin is the plasma protein that is involved in removal of zinc from intestinal-mucosal cells and subsequent transport of the metal in portal blood to the liver.     

Effect of hypophysectomy on the rates of protein synthesis and degradation in rat liver.

The effect of hypophysectomy on the protein metabolism of the liver in vivo was studied. Fractional rates of protein synthesis and degradation were determined in the livers of normal and hypophysectomized rats. Synthesis was measured after the injection of massive amounts of radioactive leucine. Degradation was estimated either as the balance between synthesis and accumulation of stable liver proteins or from the disappearance of radioactivity from the proteins previously labelled by the injection of NaH14CO3. The results indicate that: (1) hypophysectomy diminishes the capacity of the liver to synthesize proteins in vivo, mainly of those that are exported as plasma proteins; (2) livers of both normal and hypophysectomized rats show identical protein-degradation rates, whereas plasma proteins are degraded slowly after hypophysectomy.     

Comparison of protein synthesis and degradation in incubated and perfused muscle.

Rates of muscle protein synthesis and degradation measured in the perfused hindquarter were compared with those in incubated epitrochlearis muscles. With fed or starved mature rats, results without insulin treatment were identical. With insulin treatment, protein synthesis in perfused hindquarters was greater, though protein degradation was the same. Thus rates of muscle protein degradation estimated by these two methods in vitro correspond closely.     

Endurance training increases gluconeogenesis during rest and exercise in men

Plasma albumin is well known to decrease in response to inflammation. The rate of albumin synthesis from both liver and plasma was measured in vivo by use of a large dose of L-[(2)H(3)-(14)C]valine in rats injected intravenously with live Escherichia coli and in pair-fed control rats during the acute-phase period (2 days postinfection). The plasma albumin concentration was reduced by 50% in infected rats compared with pair-fed animals. Infection induced a fall in both liver albumin mRNA levels and albumin synthesis relative to total liver protein synthesis. However, absolute liver albumin synthesis rate (ASR) was not affected by infection. In plasma, albumin fractional synthesis rate was increased by 50% in infected animals compared with pair-fed animals. The albumin ASR estimated in the plasma was similar in the two groups. These results suggest that hypoalbuminemia is not due to reduced albumin synthesis during sepsis. Moreover, liver and plasma albumin ASR were similar. Therefore, albumin synthesis measured in the plasma is a good indicator of liver albumin synthesis.     

Quantitative determination of urinary N-tau-methylhistidine output as an index of myofibrillar protein degradation

N tau-Methylhistidine(3-methylhistidine) in urine of the rat is mainly derived from the degradation of actin and myosin in skeletal muscle, intestine and skin. The fractional degradation rates of the myosin-actin pools of these tissues were calculated from the time course of increase in the specific radioactivities of N tau-methylhistidine after daily administration of [methyl-14C]methionine to young adult rats under conditions of restricted food intake. The contributions to urinary excretion of N tau-methylhistidine from the three tissues were calculated from the fractional degradation rates and N tau-methylhistidine contents of the three tissues; 75.6% for skeletal muscle, 2.2% for intestine and 22.2% for skin. The results show that the skeletal muscle is the major source of urinary N-tau-methylhistidine output, but the contribution of skin is not negligible in rats. The specific radioactivity of N tau-methylhistidine in urine was much higher than that of skeletal muscle. The fractional degradation rates of myosin and actin in skeletal muscle had similar values. Although the specific radioactivities of N tau-methylhistidine in myosin and actin were very different, the mean value was similar to that in mixed skeletal muscle.