The effect of cortisol on the synthesis of rat plasma albumin, fibrinogen and transferrin.
ABSTRACT: A decrease of absolute synthesis of albumin, no change in that of fibrinogen and an increased fractional synthesis of transferrin were observed 3h after intraperitoneal administration of a pharmacological dose of 5 mg of cortisol to 220g rats in the post-absorptive state and previously kept on a diet with 40% protein. The concentration in liver of total free amino acids was practically unchanged at this time. Intraperitoneal administration of a mixture of amino acids with the cortisol raised this concentration and was accompanied by an almost complete de-repression of the synthesis of albumin, with no real effect on that of fibrinogen. In considerable contrast, in rats studied at 24h after intraperitoneal administration of cortisol, and who had been fed once in the interim (but who had received no amino acids intraperitoneally), there was a marked increase in the absolute synthesis of albumin and fibrinogen, with an increase in fractional synthesis that was less proportionately but still very significant and which included transferrin. The amino acid concentrations had risen above the supplemented values at 3h but not as much proportionately as the fractional synthesis rates, and of course not as much as the absolute synthesis rates, of albumin and fibrinogen. These time-dependent effects of cortisol suggest to us that our studies resolve the apparently conflicting results of the effect of cortisol on the synthesis of albumin reported by others.
Project description:A system using hepatocyte suspensions in vitro was developed for studying the synthesis of albumin, fibrinogen and transferrin. Conditions for optimum survival of the hepatocyte and for synthesis of these plasma proteins were defined for this system. These conditions included the use of horse serum (17.5 percent, v/v, heat-inactivated), an enriched medium (Waymouth's MB 752/1), an O2 tension of between 18.7 times 10(3) and 26.7 times 10(3) Pa and constant stirring. Albumin, fibrinogen and transferrin synthesis rates were obtained of 0.32 p 0.094(10), 0.12 p 0.030(11) and 0.097 p 0.017(10) [mean p S.D. (n)]mg/h per g of hepatocytes respectively. These rates were maintained for the first 12h of study and synthesis continued at a diminished rate up to 48h. The synthesis of albumin was decreased in a medium containing less amino acids and glucose, but that of fibrinogen was substantially unaffected. ATP concentrations up to 12h and RNA/DNA ratios up to 24h were comparable with values in vivo. The ability to study cells up to 48h permitted us to find that the addition of a mixture of hormones consisting of glucagon, cortisol, tri-iodothyronine and growth hormone enhanced fibrinogen synthesis. Addition of insulin to the above mixture resulted in increased synthesis for albumin and transferrin but not for fibrinogen.
Project description:The possibility of using two different isotopomers, for the incorporation of isotopically labeled amino acids, was explored to enable longitudinal studies of de novo synthesis of two export liver proteins, albumin and fibrinogen. The agreement of the synthesis rates between the two different labels was evaluated along with the reproducibility of repeated experiments using different time intervals. Healthy volunteers were studied in a standardized fed state. Protocol A (n = 10) involved two measurements 48 hours apart. Protocol B (n = 6) involved three measurements at baseline and five hours and then seven days after the initial measurement. De novo synthesis of albumin and fibrinogen by the incorporation of D5-phenylalanine or D8-phenylalanine were measured using the flooding dose technique. Albumin and fibrinogen were isolated from plasma using standard techniques. Fractional and absolute synthesis rates were calculated. Repeated measurements employing the two isotoptomers showed good agreement for albumin fractional synthesis rate after 48 hours (p = 0.92) and after 7 days (p = 0.99), with a coefficient of variation of 5.9% when using the same isotopic label. For fibrinogen, the coefficient of variation for the fractional synthesis rate employing the same isotopic label was 16.6%. Repeated measurements after 48 hours and seven days showed less agreement although there was no statistical difference (P = 0.32 and P = 0.30 respectively). Repeated measurement after five hours showed a statistical significant difference for the fractional synthesis rate of fibrinogen (p = 0.008) but not for albumin (p = 0.12). Repeated measurements of albumin de novo synthesis more than 48 hours apart show acceptable agreement using either one or two different isotopic labels. For fibrinogen the larger intra-individual scatter necessitates larger study groups to detect changes in longitudinal studies. Repeated measurements within 48 hours need to be validated further.
Project description:Decrease of absolute synthesis of albumin and fractional synthesis of transferrin was observed within 3h of orally administering ethanol (4ml/kg) to rats maintained on a 40%-protein diet. In contrast, absolute synthesis of fibrinogen was unaffected. With this ethanol intake, the changes in protein synthesis occurred without significant ultrastructural change in the liver. When the ethanol intake was greater (8ml/kg) ultrastructural disruption was observed. However, both the decrease of plasma protein synthesis and the ultrastructural alterations could be prevented by the simultaneous administration of a mixture of amino acids with the ethanol. The latter findings, not reported hitherto, suggest that ethanol may interfere with hepatic plasma protein synthesis and ultrastructure more through a disturbance of amino acid metabolism than through direct physical damage to the hepatocyte. An Appendix outlines the deconvolutional method used to correct for losses of labelled protein in the period during which measurements were made. The principle may also be applied to labelled plasma urea. The details of the calculations are given in a supplementary paper that has been deposited as Supplementary Publication 50007 at the National Lending Library for Science and Technology, Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1972) 126, 5.
Project description:1. Rates of synthesis of plasma albumin and fibrinogen were measured by the [(14)C]carbonate method in normal rabbits and in animals that received a single intravenous injection of Shigella endotoxin 14-48hr. earlier. 2. The accuracy of the method was improved by introducing refinements into procedures for measuring (14)C radioactivities associated with both urea and proteins that are lost from the plasma during the synthesis interval. 3. The synthesis interval (time between injecting carbonate and measuring specific radioactivities of protein guanidine carbon in plasma) can be shortened with advantage to 3-4hr. 4. Injection of endotoxin markedly decreased the fractional rate of loss in the first few hours of injected radioiodine-labelled fibrinogen and to a smaller extent of similarly labelled albumin from the plasma. The absolute rate of synthesis of fibrinogen was increased in endotoxin-treated rabbits by more than 400% compared with normal animals, and the rate of synthesis of albumin was increased by about 60%.
Project description:1. The isolated perfused rat liver was used to study degradation rates of plasma albumin, transferrin and fibrinogen. 2. Constant fractional rates were observed for all three proteins even when the albumin concentration was drastically increased by the addition of large amounts to the perfusate pool. 3. Livers taken from rats deprived of dietary protein for 14-18 days showed greatly diminished fractional catabolic rates for albumin when perfused with blood from similarly deprived animals. 4. These rates could be restored to near-normal values by adding albumin or by perfusing with blood from normally fed rats. 5. These findings are consistent with the theory of pinocytosis as a step in the degradation of plasma proteins by hepatic parenchymal cells.
Project description:A two-step method for the separation of five different plasma proteins on a preparative scale, which is capable of being extended to allow the separation of other plasma proteins, is described. The proteins separated were fibrinogen, two alpha(1)-glycoproteins, albumin and transferrin. The alpha(1)-glycoproteins were characterized in terms of electrophoretic mobility, ultracentrifugal and immunological characteristics. By using this method, it was shown that a single sample of plasma could be fractionated to yield purified proteins in sufficient quantity to simultaneously measure the synthesis of the two alpha(1)-glycoproteins, albumin and transferrin in the rat with McFarlane's technique (McFarlane, 1963; Reeve et al., 1963; McFarlane et al., 1965).
Project description:A method described by McFarlane (1963a) for the measurement of the absolute rate of synthesis of liver-made plasma proteins was used to show that, within a few hours of giving bovine growth hormone to rats, fibrinogen synthesis increased significantly without a change in albumin synthesis.
Project description:Tissue-engineered models that mimic in vivo tissue organization offer the potential of capturing complex signaling pathways in vitro. In the liver, hepatocytes and endothelial cells are closely associated but separated by the extracellular matrix of the space of Disse. This unique configuration was mimicked by embedding primary hepatocytes in collagen gel and overlaying the matrix with endothelial cells. We demonstrate that during the first few days of culture, the secretion of albumin and fibrinogen was 2-fold higher in cocultures compared to hepatocytes alone. Hepatocyte function in both cultures stabilized to a similar level during the second week, suggesting that endothelial cells can induce the early recovery of hepatocytes after isolation and seeding. Endothelial cell-conditioned medium reproduced the effect of coculture in a dose-dependent fashion, suggesting a role for endothelial cell-derived soluble factors. Endothelial cell-conditioned medium increased mRNA levels of various acute-phase proteins such as albumin, fibrinogen, transferrin, and alpha-macroglobulin in hepatocytes. Surprisingly, the effect of endothelial cell-conditioned medium was not mediated by growth factors or cytokines, or by secreted extracellular matrix, but by the release of the amino acid proline, which mediates endogenous collagen synthesis by hepatocytes. These findings suggest an important role for proline secretion by endothelial cells as a paracrine factor regulating hepatocyte function.
Project description:Hormone effects on the synthesis of alpha(1) (acute-phase) glycoprotein and of albumin by isolated rat hepatocytes in suspension were examined. Insulin, glucagon, cortisol, somatotropin (bovine growth hormone) and tri-iodothyronine were added to achieve physiological concentrations in the medium [Jeejeebhoy, Ho, Greenberg, Phillips, Bruce-Robertson & Sodtke (1975) Biochem. J.146, 141-155]. After periodic additions, there were increases (compared with values for non-hormone-treated suspensions) in the concurrent absolute syntheses of alpha(1) (acute-phase) glycoprotein and of albumin. Trends were detectable after 24h, and significant increases were demonstrated after 48h of incubation (219 and 119% respectively of control values). Manipulation of hormones, by omission from the mixture or by addition of only one or two hormones in various combinations, indicated that for alpha(1) (acute-phase) glycoprotein (which may be representative of some other acute-phase proteins), cortisol was one of the most important hormones involved in the stimulation of synthesis, with glucagon enhancing the effect of cortisol but not being stimulatory by itself. Addition of actinomycin D inhibited this stimulation, suggesting that cortisol might have acted through promotion of RNA synthesis. For albumin, cortisol alone did not stimulate synthesis, but its absence from a hormone mixture significantly decreased synthesis compared with that observed with the complete hormone mixture. Our findings support the possibility that following tissue injury, synthesis of alpha(1) (acute-phase) glycoprotein may be stimulated by the hormonal response to this injury (which response includes elevated blood concentrations of cortisol and glucagon).
Project description:1. A formula is proposed for calculating fractional synthesis rates of liver-produced plasma proteins that dispenses with urinary information or information about the size of the urea pool in the body or the fraction of urea that is endogenously catabolized. 2. Synthesis rates obtained for albumin and fibrinogen agreed well with corresponding catabolic rates for the (131)I-labelled proteins except in two of the fibrinogen measurements. 3. Significant reutilization of (14)C occurs in some animals after [(14)C]carbonate injections, giving rise to errors in the calculation of protein synthesis rates. These can best be avoided by using results obtained by injecting [(13)C]urea simultaneously. [(15)N]urea is shown not to be satisfactory for this purpose.