S. mansoni SmKI-1 Kunitz-domain: Leucine point mutation at P1 site generates enhanced neutrophil elastase inhibitory activity.

The Schistosoma mansoni SmKI-1 protein is composed of two domains: a Kunitz-type serine protease inhibitor motif (KD) and a C-terminus domain with no similarity outside the genera. Our previous work has demonstrated that KD plays an essential role in neutrophil elastase (NE) binding blockage, in neutrophil influx and as a potential anti-inflammatory molecule. In order to enhance NE blocking capacity, we analyzed the KD sequence from a structure-function point of view and designed specific point mutations in order to enhance NE affinity. We substituted the P1 site residue at the reactive site for a leucine (termed RL-KD), given its central role for KD's inhibition to NE. We have also substituted a glutamic acid that strongly interacts with the P1 residue for an alanine, to help KD to be buried on NE S1 site (termed EA-KD). KD and the mutant proteins were evaluated in silico by molecular docking to human NE, expressed in Escherichia coli and tested towards its NE inhibitory activity. Both mutated proteins presented enhanced NE inhibitory activity in vitro and RL-KD presented the best performance. We further tested RL-KD in vivo in an experimental model of monosodium urate (MSU)-induced acute arthritis. RL-KD showed reduced numbers of total cells and neutrophils in the mouse knee cavity when compared to KD. Nevertheless, both RL-KD and KD reduced mice hypernociception in a similar fashion. In summary, our results demonstrated that both mutated proteins showed enhanced NE inhibitory activity in vitro. However, RL-KD had a prominent effect in diminishing inflammatory parameters in vivo.

ß-hydroxy-ß-methyl butyrate promotes leucine metabolism and improves muscle fibre composition in growing pigs.

The aim of this study was to investigate the effects of excess leucine (Leu) vs. its metabolites α-ketoisocaproate (KIC) and ß-hydroxy-ß-methyl butyrate (HMB) on Leu metabolism, muscle fibre composition and muscle growth in growing pigs. Thirty-two pigs with a similar initial weight (9.55 ± 0.19 kg) were fed 1 of 4 diets for 45 days: basal diet, basal diet + 1.25% L-Leu, basal diet + 1.25% KIC-Ca, basal diet + 0.62% HMB-Ca. Results indicated that relative to the basal diet and HMB groups, Leu and KIC groups exhibited increased Leu concentrations and decreased concentrations of isoleucine, valine and EAAs in selected muscle (p < 0.05) and had lower mRNA levels of MyHC I and higher expression of MyHC IIx/IIb (p < 0.05), and there was no significant difference between the basal and HMB-supplemented groups. Moreover, the mRNA expression levels of AMPKα and UCP3 were higher but the myostatin mRNA levels were lower in the soleus muscle of the HMB group than those from other groups (p < 0.05). These findings demonstrated that doubling dietary Leu content exerted growth-depressing effects in growing pigs; dietary KIC supplementation induced muscular branched-chain amino acid imbalance and promoted muscle toward a more glycolytic phenotype; while dietary HMB supplementation promoted the generation of more oxidative muscle types and increased muscle growth specially in oxidative skeletal muscle, and these effects of HMB might be associated with the AMPKα-Sirt1-PGC-1α axis and mitochondrial biogenesis.

Celastrofurans A-G: Dihydro-ß-agarofurans from the Australian Rainforest Vine Celastrus subspicata and Their Inhibitory Effect on Leucine Transport in Prostate Cancer Cells.

Seven new dihydro-ß-agarofurans, celastrofurans A-G (1-7), along with two known secondary metabolites, 9ß-benzoyloxy-1α-furoyloxydihydro-ß-agarofuran (8) and (1R,2R,4R,5S,7R,9S,10R)-2-acetoxy-9-benzoyloxy-1-furoyloxydihydro-ß-agarofuran (9), were obtained from the leaves of the Australian rainforest vine, Celastrus subspicata. The structures of the new compounds were determined by detailed spectroscopic (1D/2D NMR) and MS data analysis. The absolute configurations of compounds 1-4 were defined by ECD and single-crystal X-ray diffraction studies. All compounds were found to exhibit inhibitory activity on leucine transport in the human prostate cancer cell line LNCaP with IC50 values ranging from 7.0 to 98.9 µM. Dihydro-ß-agarofurans 1-9 showed better potency than the L-type amino acid transporter (LAT) family inhibitor, 2-aminobicyclo[2.2.1]-heptane-2-carboxylic acid (BCH).

Specificity of the volume-activated amino acid efflux pathway in cultured human breast cancer cells.

It has been shown that cell swelling stimulates the efflux of taurine from MCF-7 and MDA-MB-231 cells via a pathway which has channel-like properties. The purpose of this study was to examine the specificity of the volume-activated taurine efflux pathway in both cell lines. A hyposmotic shock increased the efflux of glycine, L-alanine, AIB (α-aminoisobutyric acid), D-aspartate but not L-leucine from MDA-MB-231 and MCF-7 cells. It was evident that the time course of activation/inactivation of those amino acids whose efflux was affected by cell swelling was similar to that of volume-activated taurine efflux. The effect of exogenous ATP on swelling-induced glycine, AIB and D-aspartate efflux from MDA-MB-231 cells was similar to that found on taurine efflux. In addition, volume-activated AIB efflux from MDA-MB-231 cells, like that of swelling-induced taurine efflux, was inhibited by diiodosalicylate. Tamoxifen inhibited volume-activated taurine release from both MDA-MB-231 and MCF-7 cells. The results suggest that neutral and anionic α-amino acids are able to utilize the volume-activated taurine efflux pathway in both cell lines. The effect of tamoxifen on breast cancer growth may, in part, be related to perturbations in cell volume regulation.

Leucine induces phosphorylation and activation of p70S6K in cortical neurons via the system L amino acid transporter.

p70S6 kinase is a multipotent kinase that phosphorylates substrates in response to extracellular stimuli. This kinase activity inhibits apoptosis, regulates cell size and controls translation. In the CNS, p70S6K also participates in synaptic plasticity. In this study, we report that leucine, a branched-chain amino acid, induces phosphorylation and activation of p70S6 kinase in cortical neurons. Leucine also induces phosphorylation of S6 protein, a substrate of p70S6K. These effects of leucine are completely inhibited by rapamycin, consistent with mammalian target of rapamycin mediating p70S6 phosphorylation. Finally, we demonstrate that the action of leucine on cortical neurons is mediated by the system L amino acid transporter. Neurons express components of system L amino acid transporter LAT1, LAT2, and CD98. Leucine uptake and its effect on p70S6 kinase are both inhibited by a specific inhibitor of system L amino acid transporter. We propose that leucine plays important roles in regulating signaling by p70S6 kinase by acting as an intercellular communicator in the CNS.

Effect of casein and propionate supply on mammary protein metabolism in lactating dairy cows.

The effects of casein (CN) and propionate (C3) on mammary AA metabolism were determined in 3 multiparous Holstein cows fitted with both duodenal and ruminal cannulas and used in a replicated Youden square with six 14-d periods. Casein (743 g/d in the duodenum) and C3 (1,041 g/d in the rumen) infusions were tested in a factorial arrangement. For each period, L-[1-(13)C]Leu (d 11) and NaH[13C]O3 (d 13) were infused into a jugular vein, and blood samples were taken from the carotid artery and the mammary vein to determine Leu kinetics and net uptake of AA. Both CN and C3 treatments separately increased milk protein concentration and yield. With CN there was a general response in mammary protein metabolism, involving increases in Leu net uptake (30%), the uptake:output ratio (8%), protein synthesis (11%), secretion in milk protein (21%), and oxidation (259%). In contrast, C3 treatments tended to increase only Leu in milk protein (7%) and, when in combination with CN, to reduce Leu used for protein synthesis (5%). Across all treatments, most Leu uptake by the mammary gland was accounted for as Leu in milk or oxidized, and the Leu balance was therefore achieved without involvement of either net peptide use or production. Mammary uptake of group 1 AA increased to match milk output with all infusions. In contrast, mammary uptake of group 2 AA exceeded output to a greater extent with CN than with C3 infusions, whereas the increment in uptake of group 3 AA increased with C3 treatments. Overall, these data suggest that different mechanisms operate to improve milk protein production when either protein or energy is supplied.

Protease inhibitor therapy improves protein catabolism in prepubertal children with HIV infection.

UNLABELLED: Past studies in adults have reported that loss of lean tissue mass (LTM) is associated with accelerated rates of protein catabolism. To date, studies of protein kinetics from pediatric patients infected by HIV have not been published; however, poor linear growth and weight loss are well-documented. The first aim of this study was to test the hypothesis that protein catabolism is high in pediatric patients with HIV. Protease inhibitors (PI) have proven to be effective therapy for pediatric HIV patients. One action of these drugs is that of lowering the viral burden, and several studies suggest that these drugs result in increased growth and weight velocity. Our second aim was to determine whether PI therapy improves protein catabolism. METHODS: We studied eight children infected with HIV (ages 2.9-6.2 years, Tanner stage I, CD4 counts 100,000-300,000, 5 F/3 M) and eight healthy age- and gender-matched controls. Measures of protein turnover were conducted using the stable isotope [1-(13C)]leucine. Body composition was measured by dual X-ray absorptiometry (DXA) scan for determination of LTM, and indirect calorimetry for measurement of resting energy expenditure. Children with HIV infection were studied at baseline and after 6 weeks of PI therapy; control children were studied only once. RESULTS: Protein catabolism, represented as leucine rate of appearance (Ra) in the fasted state, was higher in the HIV-infected children at baseline compared to control children. After 6 weeks of PI therapy, leucine Ra decreased, but not to the range found in control children. Leucine Ra correlated with viral burden. LTM significantly improved in all patients. CONCLUSION: These results suggest that similar to HIV-infected adults, HIV-infected children have higher than normal protein catabolism. Furthermore, our measures suggest that short-term PI therapy results in improved protein catabolism and LTM.

Amino acids in transmembrane domain two influence anesthetic enhancement of serotonin-3A receptor function.

Alcohols and volatile anesthetics affect the function of members of the nicotinic acetylcholine (nACh) superfamily of receptors. Studies on glycine and GABA(A) receptors implicate amino acid residues within transmembrane (TM) regions two and three of these receptors as critical for alcohol and anesthetic enhancement of receptor function. The serotonin-3 (5-HT(3)) receptor is a member of the nicotinic acetylcholine receptor superfamily, sharing sequence and structural homology with the other members. We tested the hypothesis that amino acids of the 5-HT(3) receptor homologous to those shown to affect alcohol and anesthetic potentiation in GABA(A) and glycine receptors also determine the effects of these compounds on the 5-HT(3) receptor. Six 5-HT(3A) mutant cDNAs were generated by site-directed mutagenesis of two amino acids, phenylalanine-269 (14') and lecucine-270 (15') in transmembrane domain two (TM2). When assayed electrophysiologically in Xenopus oocytes, wild-type 5-HT(3) receptors exhibit enhancement of function by enflurane, halothane, isoflurane, chloroform and ethanol, but not by decanol and propofol. Mutations in transmembrane domain two markedly affected alcohol and anesthetic enhancement of 5-HT(3) receptor function. Some mutations had differential effects on the abilities of the isomers enflurane and isoflurane to potentiate 5-HT(3) receptor function.

Effects of tanshinone VI on the hypertrophy of cardiac myocytes and fibrosis of cardiac fibroblasts of neonatal rats.

The possible effects of tanshinone VI (tsh), a diterpene from the root of Tan-Shen (Salvia miltiorrhiza, Bunge (Labiatae)) on hypertrophy and fibrosis in cultured neonatal rat cardiac myocytes and fibroblasts were examined. Tsh had no significant effect on protein synthesis, which was evaluated by [3H]-leucine incorporation into the acid insoluble fraction in the cells, in the absence of stimulatory factors in cardiac myocytes. The amount of protein produced in cardiac myocytes was increased by 10(-8) M endothelin-1 (ET-1), 10(-6) M phenylephrine (PE), or 10(-8) M insulin-like growth factor-1 (IGF-1), suggesting that hypertrophy of cardiac myocytes in vitro was induced by these factors. The ET-1-, PE-, or IGF-1-induced increase in protein synthesis was attenuated by treatment with 10(-5) M tsh. Treatment with 10(-5) M tsh significantly decreased the synthesis of collagen by cardiac fibroblasts, which was evaluated by [3H]-proline incorpolation into acid-insoluble fraction of the fiblobrasts, in the absence of stimulatory factors for the production. Fetal bovine serum (FBS) or IGF-1 increased collagen synthesis in a concentration-dependent manner. The increase at 5% FBS or 10(-8) M IGF-1 was inhibited by 10(-5) M tsh. Fibroblast-conditioned medium (FB-CM) increased protein synthesis in cardiac myocytes in a concentration-dependent manner (10; - 100 %). Tsh attenuated the FB-CM-induced increase in protein synthesis by cardiac myocytes. These results show that tsh may attenuate the humoral factor-induced hypertrophy of cardiac myocytes and fibrosis of cardiac fibroblasts. The findings suggest that tsh may improve the development of cardiac remodeling under pathophysiological conditions. Abbreviations. ANP:atrial natriuretic peptide DMEM:Dulbecco-modified Eagle's medium ET-1:endothelin-1 FB-CM:fibroblast-conditioned medium FBS:fetal bovine serum IGF-1:insulin-like growth factor-1 PE:phenylephrine tsh:tanshinone VI

System y+L-like activities account for high and low amino-acid transport phenotypes in chicken erythrocytes.

The functional properties of the transport of lysine across the chicken erythrocyte membrane were investigated. The animal population studied (male Leghorn chickens, 6-14 weeks old) was found to consist of two groups presenting either low (LT, 19 individuals) or high transport rates (HT, 20 individuals). The rates of influx in the two groups, measured at a concentration of l-lysine of 1 microm, differed by a factor of 34. The transport activities observed in LT and HT erythrocytes were compatible with the general features of system y+L, but showed some differences in specificity. The transporter in the LT group was found to bind l-lysine, l-leucine, l-methionine and l-glutamine with high affinity, in the presence of sodium, as described for system y+L in human erythrocytes. The activity present in HT erythrocytes exhibited a much lower affinity for l-leucine, but was able to interact strongly with l-glutamine and l-methionine. The specificity pattern of the HT transporter, has not been described in other cell types. In other respects, the properties of the two systems were similar. Sodium replacement with potassium, drastically reduced the affinity for l-leucine, without affecting lysine transport. Both transporters function as tightly coupled exchangers, are inactivated by p-chloromercuribenzene sulfonate and resistant to N-ethylmaleimide. These findings explain previous results obtained in selective breeding experiments of chicken with high and low amino-acid transport activity.

Reactive oxygen species mediate amplitude-dependent hypertrophic and apoptotic responses to mechanical stretch in cardiac myocytes.

Oxidative stress stimulates both growth and apoptosis in cardiac myocytes in vitro. We investigated whether oxidative stress mediates hypertrophy and apoptosis in cyclically stretched ventricular myocytes. Neonatal rat ventricular myocytes cultured on laminin-coated silastic membranes were stretched cyclically (1 Hz) at low (nominal 5%) and high (nominal 25%) amplitudes for 24 hours. Stretch caused a graded increase in superoxide anion production as assessed by superoxide dismutase (SOD)-inhibitable cytochrome c reduction or electron paramagnetic resonance spectroscopy. The role of reactive oxygen species (ROS) was assessed using the cell-permeable SOD/catalase mimetics Mn(II/III)tetrakis(1-methyl-4-peridyl) (MnTMPyP) and EUK-8. Stretch-induced increases in protein synthesis ((3)H-leucine incorporation) and cellular protein content were completely inhibited by MnTMPyP (0.05 mmol/L) at both low and high amplitudes of stretch. In contrast, while MnTMPyP inhibited basal atrial natriuretic factor (ANF) mRNA expression, the stretch-induced increase in ANF mRNA expression was not inhibited by MnTMPyP. In contrast to hypertrophy, only high-amplitude stretch increased myocyte apoptosis, as reflected by increased DNA fragmentation on gel electrophoresis and an approximately 3-fold increase in the number of TUNEL-positive myocytes. Similarly, only high-amplitude stretch increased the expression of bax mRNA. Myocyte apoptosis and bax expression stimulated by high-amplitude stretch were inhibited by MnTMPyP. Both low- and high-amplitude stretch caused rapid phosphorylation of ERK1/2, while high-, but not low-, amplitude stretch caused phosphorylation of JNKs. Activation of both ERK1/2 and JNKs was ROS-dependent. Thus, cyclic strain causes an amplitude-related increase in ROS, associated with differential activation of kinases and induction of hypertrophic and apoptotic phenotypes.

Identification of a novel inhibitor of LPS-induced TNF-alpha production with antiproliferative activity in monocyte/macrophages.

An isoquinoline derivative, 5-methyl-7,8-dimethoxy-1-phenylpyrazolo[5,4-c]isoquinoline (compound 1), was identified as a novel inhibitor of LPS-induced TNF-alpha production by cell-based screening. Compound 1 suppressed LPS-induced TNF-alpha production in RAW264.7 cells and murine peritoneal macrophages in a dose-dependent manner similar to SB203580, known as a specific inhibitor of p38 MAPK. It also inhibited an LPS-induced increase in serum TNF-alpha in a mouse endotoxic shock model with an ED(50) of approximately 10 mg/kg. Compound 1 had little effect on the incorporation of [3H]-leucine into the cells, while it suppressed LPS-induced TNF-alpha mRNA levels in RAW264.7 cells. The results indicate that suppression of TNF-alpha production was not a result of nonspecific inhibition of de novo translation but was based on the decreased TNF-alpha mRNA levels. The in vitro kinase assay revealed that compound 1 did not strongly inhibit p38 MAPK activity, its potency being much lower than that of SB203580, suggesting that the TNF-alpha-suppressive action of compound 1 cannot be attributed to the inhibition of p38 MAPK. Furthermore, in contrast to SB203580, it significantly inhibited the growth of RAW264.7 and THP-1 cells in a cytostatic manner. Compound 1 is likely to have antiinflammatory and antiproliferative effects by acting on some molecule other than p38 MAPK that contributes to both LPS-induced TNF-alpha production and the cell growth of monocyte/macrophages.

Overexpression of 12-lipoxygenase causes cardiac fibroblast cell growth.

Evidence suggests that leukocyte type 12-lipoxygenase (12-LO) plays an important role in cell growth. However, the role of 12-LO in cardiac cell growth has not been tested. We have now stably overexpressed 12-LO cDNA in rat fetal cardiac fibroblasts to evaluate the role of the 12-LO pathway in cardiac cell growth. Overexpression of 12-LO increased cell [(3)H]leucine incorporation by 2.1+/-0.1-fold (P<0.01) and cell protein content by 2.2+/-0. 3-fold (P<0.01) over mock-transfected cells. These findings were confirmed in additional clones. Baicalein, a 12-LO enzyme inhibitor, dose-dependently inhibited serum-induced leucine incorporation in cardiac fibroblast cells as well as partially inhibited leucine incorporation in cells overexpressing 12-LO. 12-LO overexpression also caused cell [(3)H]thymidine incorporation to increase by 3.4+/-0.3-fold (P<0.01). Cell flow cytometry analysis showed that the size of 12-LO-overexpressing cells was markedly enlarged compared with that of mock-transfected cells. The fibronectin content of the 12-LO-overexpressing cardiac fibroblasts was also significantly increased. We next evaluated the effects of 12-LO RNA overexpression on kinase pathways linked to cellular growth. The overexpression of 12-LO enhanced extracellular signal-regulated kinase activity (4. 1+/-0.5-fold), c-Jun NH(2)-terminal kinase activity (2.9+/-0.5-fold), and p38 mitogen-activated protein kinase activity (2.2+/-0.3-fold). Pretreatment with SB202190 (100 nmol/L), a specific inhibitor of p38, prevented the increases in protein content of 12-LO-overexpressing cardiac fibroblast cells. These data clearly demonstrate that the overexpression of 12-LO causes cell growth of cardiac fibroblasts, thus supporting the role of 12-LO as a novel growth-promoting pathway in the heart.

Effect of alanyl-glutamine on leucine and protein metabolism in endotoxemic rats.

BACKGROUND: Branched-chain amino acids (BCAA; valine, leucine, and isoleucine) have a regulatory effect on protein metabolism and are the main donor for synthesis of alanine and glutamine in the skeletal muscle. This study was performed to investigate whether exogenous alanine or glutamine would affect leucine and protein metabolism in intact and endotoxemic rats. METHODS: Rats were injected with endotoxin of Salmonella enteritidis or saline. Thirty minutes later, the effects of endotoxemia and L-alanyl-L-glutamine (AG) on leucine and protein metabolism were evaluated using a primed constant infusion of [1-14C]leucine, endotoxin, and AG (200 mg/mL) solution or an infusion of [1-14C]leucine without endotoxin or AG. The specificity of the effect of exogenous alanine and glutamine was evaluated by a single infusion of alanine, glutamine, and glycine in a separate study. RESULTS: Endotoxin treatment induced more negative net protein balance caused mainly by an increase in whole-body proteolysis. Protein synthesis increased in kidneys, colon, and spleen, while a decrease was observed in skeletal muscle. The impressive effects of AG were the decrease in plasma branched-chain amino acid (BCAA) levels, decrease in leucine oxidized fraction, and improvement of protein balance associated with a decrease in whole-body proteolysis. Similar changes in leucine and protein metabolism were induced by infusion of alanine or glutamine but not by infusion of glycine. CONCLUSIONS: IV administration of alanine or glutamine improves protein balance and decreases leucine oxidized fraction in postabsorptive state and in endotoxemia. Decreased proteolysis is the main cause of decreased plasma BCAA levels after AG treatment.

Differential regulation of amino acid exchange and protein dynamics across splanchnic and skeletal muscle beds by insulin in healthy human subjects.

To define the mechanism of insulin's anticatabolic action, the effects of three different dosages of insulin (0.25, 0.5, and 1.0 mU x kg(-1) x min(-1)) versus saline on protein dynamics across splanchnic and skeletal muscle (leg) beds were determined using stable isotopes of phenylalanine, tyrosine, and leucine in 24 healthy subjects. After an overnight fast, protein breakdown in muscle exceeded protein synthesis, causing a net release of amino acids from muscle bed, while in the splanchnic bed protein synthesis exceeded protein breakdown, resulting in a net uptake of these amino acids. Insulin decreased (P < 0.003) muscle protein breakdown in a dose-dependent manner with no effect on muscle protein synthesis, thus decreasing the net amino acid release from the muscle bed. In contrast, insulin decreased protein synthesis (P < 0.03) in the splanchnic region with no effect on protein breakdown, thereby decreasing the net uptake of the amino acids. In addition, insulin also decreased (P < 0.001) leucine nitrogen flux substantially more than leucine carbon flux, indicating increased leucine transamination (an important biochemical process for nitrogen transfer between amino acids and across the organs), in a dose-dependent manner, with the magnitude of effect being greater on skeletal muscle than on the splanchnic bed. In conclusion, muscle is in a catabolic state in human subjects after an overnight fast and provides amino acids for synthesis of essential proteins in the splanchnic bed. Insulin achieves amino acid balance across splanchnic and skeletal muscle beds through its differential effects on protein dynamics in these tissue beds.

DNA methylation by wheat cytosine DNA methyltransferase: modulation by protease inhibitor E-64.

Cytosine DNA methyltransferase isolated from wheat seedlings and purified in the presence of metalloprotease and serine protease inhibitors has molecular mass and specific activity equal to about 85 kDa and 250 units/mg protein, respectively. Apparent K(m) for AdoMet and [I]50 for AdoHcy values are about 6 microM and 12 microM, respectively. The enzyme is active in wide pH range (pH 5.5-8.5) and is inhibited by NaCl. The enzyme rapidly loses its methyltransferase activity in the absence of substrates. Using the cysteine protease inhibitor E-64 it has been shown that rapid enzyme inactivation is caused by disappearance of essential enzyme SH-groups but is not due to proteolytic enzyme cleavage.

Hyperglucagonemia and the immediate fate of dietary leucine: a kinetic study in humans.

The possible role of glucagon in determining the fate of dietary absorbed amino acids within the splanchnic bed was investigated in five healthy male volunteers. A kinetic study was performed involving a continuous 240-minute infusion of L-[5,5,5-2H3]leucine and D-[6,6-2H2]glucose by vein, while L-[1-13C]leucine was infused by a feeding tube into the duodenum (intragut [i.g.]) along with a constant intravenous (i.v.) infusion of somatotropin release-inhibitory factor (SRIF) combined with insulin, growth hormone, and glucagon. In random order, glucagon was infused at a rate of 0.4 ng x kg(-1) x min(-1) in one experiment and 1.2 ng x kg(-1) x min(-1) in the other experiment, while insulin and growth hormone were kept at constant serum levels, respectively, 37+/-13 pmol x L(-1) and 5+/-0.2 microg x L(-1). The diet was provided as an L-amino acid solution including 60 micromol x kg(-1) x h(-1) leucine without fat and carbohydrate. During the higher rate of glucagon infusion, there was an increase in plasma glucagon and glucose concentrations, glucose flux, and net dietary leucine release into the periphery from the splanchnic bed. Splanchnic removal and uptake of leucine were decreased with increased glucagon infusion. There were no statistical differences in the plasma leucine level and i.v. and i.g. leucine fluxes at the two glucagon levels, although leucine metabolic clearance increased (0.74 v 0.85 L x kg(-1) x h(-1), P=.08) in the case of glucagon excess. Plasma glucose increased with glucagon excess and was negatively correlated (P < .05) with the plasma leucine level (r=-.348) and i.v. (r=-.459) or i.g. (r=-.359) leucine fluxes. The negative correlation between plasma glucagon and leucine levels was also significant (r=-.684). No significant correlation was found between dietary leucine splanchnic removal and glucose, glucagon, or leucine plasma concentrations. We conclude that glucagon in excess has only a small quantitative effect on the overall handling of dietary leucine, and hypothesize that more leucine is exported to the peripheral tissues under these hormonal conditions.

[The effect of chloditan on synthetic processes in the adrenal gland cells of newborn piglets]. / Vplyv khlodytanu na syntetychni protsesy v klitynakh nadnyrkovykh zaloz novonarodzhenykh porosiat.

In experiments with a primary culture of adrenal cells, it has been studied the influence of chloditane, as a specific inhibitor of adrenocortical function, on the synthetic processes in the newborn pig adrenal cells, whose organ and cell cultures are more and more used as transplantation material for the treatment of adrenal insufficiency. It has been shown that incubation of adrenocorticocytes with chloditane (final concentration in culture medium: 1 to 10,000 ng/ml) during 24 hours had a marked negative dose-dependent effect on the indices of inclusion of 3H-thymidine and 3H-uridine into the nucleic acids. In the presence of a maximum chloditane concentration, inclusion made, respectively, 16.9 and 46.0% from control. A trustworthy effect of chloditane on the indices of 3H-leucine inclusion (protein synthesis) has been established only for concentration 10 to 100 ng/ml and made near 70% from control. It has been discussed a possible mechanism of chloditane action on the synthetic processes in adrenal cells.

Evidence for acute stimulation of fibrinogen production by glucagon in humans.

Fibrinogen, an acute-phase protein, and glucagon, a stress hormone, are often elevated in many conditions of physical and metabolic stress, including uncontrolled diabetes. However, the possible mechanisms for this association are poorly known. We have studied the acute effects of selective hyperglucagonemia (raised from -200 to -350 pg/ml for 3 h) on fibrinogen fractional secretion rate (FSR) in eight normal subjects during infusion of somatostatin and replacement doses of insulin, glucagon, and growth hormone. Fibrinogen FSR was evaluated by precursor-product relationships using either Phe (n = 8) or Leu (n = 2) tracers. Hyperglucagonemia did not change either plasma Phe or Tyr specific activity. After hyperglucagonemia, fibrinogen FSR increased by approximately 65% (from 12.9 +/- 3.6 to 21.5 +/- 6.1% per day, P < 0.025) using plasma Phe specific activity as the precursor pool. FSR increased by approximately 80% (from 16.6 +/- 4.8 to 29.4 +/- 8.8% per day, P < 0.025) if plasma Phe specific activity was corrected for the ketoisocaproate/Leu enrichment (or specific activity) ratio to obtain an approximate estimate of intrahepatic Phe specific activity. FSR increased by approximately 60% when using plasma Tyr specific activity as precursor pool (n = 8) (P < 0.05), as well as when using the Leu tracer precursor-product relationship (n = 2). In conclusion, selective hyperglucagonemia for approximately 3 h acutely stimulated fibrinogen FSR using a Phe tracer method. Thus, glucagon may be involved in the increase of fibrinogen concentration and FSR observed under stressed or pathologic conditions.

Insulin-like growth factor-I and insulin reduce leucine flux and oxidation in conscious tumor necrosis factor-infused dogs.

BACKGROUND: We have tested the hypothesis that insulin-like growth factor-I (IGF-I) or insulin can prevent the protein catabolic effects of tumor necrosis factor (TNF). METHODS: After a 2-hour basal period TNF was infused (prime, 2.5 micrograms.kg-1; constant, 31.25 ng.kg-1.min-1) for 4 hours into conscious dogs to create the catabolic state. After 2 hours of TNF infusion either recombinant human IGF-I (n = 5) or recombinant human insulin (n = 5) was infused for an additional 2 hours. A third group (n = 5) received TNF alone for 4 hours. RESULTS: TNF infusion caused an increase in both glucose production, reflected by [6,6-d2]glucose tracer data, and net protein catabolism, reflected by both [1-13C]leucine and [15N2]urea tracer methods. IGF-I and insulin both significantly reduced the rates of appearance of leucine and leucine oxidation to a similar extent, resulting in the significant decrease in net protein catabolism. CONCLUSIONS: IGF-I and insulin can ameliorate the catabolic effects of TNF on protein and glucose metabolism equally effectively, although more IGF-I is required on a molar basis.