Index Of Protein Synthesis Research Articles

Glutathione Supplementation of Parenteral Nutrition Prevents Oxidative Stress and Sustains Protein Synthesis in Guinea Pig Model.

Peroxides contaminating parenteral nutrition (PN) limit the use of methionine as a precursor of cysteine. Thus, PN causes a cysteine deficiency, characterized by low levels of glutathione, the main molecule used in peroxide detoxification, and limited growth in individuals receiving long-term PN compared to the average population. We hypothesize that glutathione supplementation in PN can be used as a pro-cysteine that improves glutathione levels and protein synthesis and reduces oxidative stress caused by PN. One-month-old guinea pigs (7–8 per group) were used to compare glutathione-enriched to a non-enriched PN, animals on enteral nutrition were used as a reference. PN: Dextrose, amino acids (Primene), lipid emulsion (Intralipid), multivitamins, electrolytes; five-day infusion. Glutathione (GSH, GSSG, redox potential) and the incorporation of radioactive leucine into the protein fraction (protein synthesis index) were measured in the blood, lungs, liver, and gastrocnemius muscle. Data were analysed by ANOVA; p < 0.05 was considered significant. The addition of glutathione to PN prevented the PN-induced oxidative stress in the lungs and muscles and supported protein synthesis in liver and muscles. The results potentially support the recommendation to add glutathione to the PN and demonstrate that glutathione could act as a biologically available cysteine precursor.

English

One of the attributes of amino acid lysine is to manufacture muscle protein, as it is a physiologically essential amino acid for immunity, development and production in birds. This study evaluates the influence of several levels of the essential amino acid lysine for chicks, with age of about 504 hours, maintained at a temperature of approximately 29 degrees Celsius. The basal diet contained 21.0% crude protein and 3,000 kcal of energy of biological transformations/kg. This formula was supplemented with levorotatory-lysine acid monohydrochloride, resulting in diets with 1.040 to 1.280% of total lysine for five experimental treatments. The treatments were found to have a mathematical expression squared influence on animals’ weight gain, conversion of diet, total liver mass and protein synthesis index. The effect of the five experimental treatments was not observed in diet consumption, fat synthesis index, absolute weights of intestines and heart, and percentage of liver mass. It was concluded that the total lysine requirements for chicks aged up to 504 h were 1.240%. Key words: Thermal environment, dietary lysine, growth performance, rearing setting.

Determinants of skeletal muscle protein turnover following severe burn trauma in children

Burns remain the fifth cause of non-fatal pediatric injuries globally, with muscle cachexia being a hallmark of the stress response to burns. Burn-induced muscle wasting is associated with morbidity, yet the determinants of muscle protein catabolism in response to burn trauma remains unclear. Our objective was to determine the effect of patient and injury characteristics on muscle protein kinetics in burn patients. This retrospective, observational study was performed using protein kinetic data from pediatric patients who had severe burns (>30% of the total body surface area burned) and underwent cross-limb stable isotope infusions between 1999 and 2008 as part of prospective clinical trials. Mixed multiple regression models were used to assess associations between patient/injury characteristics and muscle protein fractional synthesis rate (FSR), net balance (NB), and rates of phenylalanine appearance (Ra; index of protein breakdown) and disappearance (Rd; index of protein synthesis) across the leg. A total of 268 patients who underwent 499 studies were analyzed. Increasing time post injury was associated with greater FSR (p<0.001) and NB (p=0.01). Males were more catabolic than females (as indicated by lower NB, p=0.04 and greater Ra, p=0.008), a consequence of higher protein breakdown rather than lower synthesis. Increasing burn size was associated with higher protein synthesis rate (as indicated by higher FSR, p=0.019) and higher protein breakdown rates (as indicated by greater Ra, p=0.001). FSR was negatively associated with age (p<0.001). Data from this large patient cohort show that injury severity, sex, and time post injury influence skeletal muscle wasting in burned children. These findings suggest that individual patient characteristics should be considered when devising therapies to improve the acute care and rehabilitation of burn survivors.

ВПЛИВ РЕГУЛЯТОРУ РОСТУ ЗА УМОВ ДІЇ АНТРОПОГЕННИХ ЧИННИКІВ ДОВКІЛЛЯ

&lt;p&gt;The paper analyses the general (GА, nM pyruvic acid/ml∙second) and specific (SA, nM pyruvic acid /mg second) transferase enzyme activity of protein metabolism (Аlanine aminotransferase ALT, EC 2.6.1.2, and Аspartate aminotransferase, AST, EC 2.6.1.1) in &lt;em&gt;Salix alba&lt;/em&gt; L. leaves, that planted on the banks of Mokra Sura River (anthropogenic polluted, increased level of salinity) and Shpakova River (relatively clean, control) which are parts of Dnipro River Basin of Steppe Dnipro Region. We used the plant growth regulator “Kornevin” in order to accelerate rooting and reducing of exogenous pressures on the plant. We registered the Aminotransferase nonspecific reaction towards anthropogenic pressure, which was associated with the formation of non-specific mechanisms of adaptation to support the homeostasis. We revealed the significant differences between experiment and control in index of protein synthesis and metabolism depending on the conditions of growth and development. Protective and leveling effects of growth regulator have been proved. The advisability of using the "Kornevin" as an adaptogene and a protector in variable environmental conditions have been indicated.&lt;/p&gt;&lt;p&gt;&lt;em&gt;Salix alba L., increased level of salinity, growth regulators, alaninaminotransferase, aspartataminotransferase, adaptogene, anthropogenic factors&lt;/em&gt;&lt;em&gt;&lt;/em&gt;&lt;/p&gt;

Formoterol, a Highly β2-Selective Agonist, Induces Gender-Dimorphic Whole Body Leucine Metabolism in Humans

ObjectiveFormoterol is a β2-selective agonist that enhances protein anabolism in rodents. Whether formoterol imparts anabolic benefits in humans is unknown. The objective of the study was to investigate the effects of formoterol on whole body protein rates of turnover, oxidative loss and synthesis. DesignOpen label intervention study. PatientsFifteen healthy adults (8 men). MeasurementsVolunteers were treated with oral formoterol 160μg/day for one week. Changes in leucine turnover (LRa; index of protein breakdown), oxidation (Lox; irreversible protein loss) and incorporation into protein (LIP; index of protein synthesis) were assessed using the whole body 1-[13C]leucine turnover technique before/after treatment. ResultsLRa, Lox and LIP correlated significantly with lean body mass (LBM). LRa, adjusted for LBM was significantly higher (P<0.05, 160±6 vs 109±3μmol/min) in men but not fractional Lox and LIP (expressed as a proportion of LRa). Formoterol reduced LRa (−9±4%) in men but stimulated LRa (9±3%) in women. Formoterol significantly reduced (P<0.05) fractional Lox, an effect greater in women (−4±1 vs −1±1 %). It stimulated fractional LIP in women (∆4±1%, P<0.05) but not in men (∆1±1%). Formoterol induced an absolute anabolic effect that was greater in women (30 vs 8%). Heart rate, systolic and diastolic blood pressures were unaffected. ConclusionIn a therapeutic dose, formoterol stimulates protein anabolism in humans. It induced gender-dimorphic effects on protein turnover and on the partitioning of amino acids from oxidative loss toward protein synthesis, effects that are greater in women than in men. Formoterol holds promise as a treatment for sarcopenia.

Growth hormone enhances fat-free mass and glutamine availability in patients with short-bowel syndrome: an ancillary double-blind, randomized crossover study , ,

Benefits of recombinant human growth hormone (rhGH) alone or combined with glutamine in patients with intestinal failure because of short-bowel syndrome remain controversial. We explored effects of rhGH on whole-body protein metabolism in patients with short-bowel syndrome with intestinal failure (SBS-IF) to gain insight into its mechanism of action. Eight stable hyperphagic patients with severe SBS-IF received, in a double-blind, randomized crossover study, low-dose rhGH (0.05 mg · kg⁻¹ · d⁻¹) and a placebo for two 3-wk periods. Leucine and glutamine kinetics under fasting and fed conditions, fat-free mass (FFM), and serum insulin were determined on the final day of each treatment. rhGH increased FFM and nonoxidative leucine disposal (NOLD; an index of protein synthesis) (P < 0.02), whereas FFM and NOLD were correlated in the fed state (r = 0.81, P = 0.015). With rhGH administration, leucine release from protein breakdown (an index of proteolysis) decreased in the fed compared with fasting states (P = 0.012), which was not observed with the placebo. However, the fast-to-fed difference in leucine release from protein breakdown was not significantly different between rhGH and placebo (P = 0.093). With rhGH, the intestinal absorption of leucine and glutamine increased (P = 0.036) and correlated with serum insulin (r = 0.91, P = 0.002). rhGH increased glutamine de novo synthesis (P < 0.02) and plasma concentrations (P < 0.03) in both fasting and fed states. In SBS-IF patients, feeding fails to decrease proteolysis in contrast to what is physiologically observed in healthy subjects. rhGH enhances FFM through the stimulation of protein synthesis and might decrease proteolysis in response to feeding. Improvements in de novo synthesis and intestinal absorption increase glutamine availability over the physiologic range, suggesting that beneficial effects of rhGH in hyperphagic patients might be achieved without glutamine supplementation.

Impaired muscle protein anabolic response to insulin and amino acids in heart failure patients: relationship with markers of immune activation

Patients with chronic HF (heart failure) experience muscle atrophy during the course of the disease. The mechanisms underlying muscle atrophy in HF, however, are not understood. Thus we evaluated leg phenylalanine balance and kinetics in HF patients and controls following a brief fast (24 h) and under euglycaemic-hyperinsulinaemic-hyperaminoacidaemic conditions to determine whether HF increases muscle protein catabolism in response to nutritional deprivation and/or diminishes the anabolic response to meal-related stimuli (insulin and amino acids) and whether alterations in protein metabolism correlate to circulating cytokine levels. No differences in phenylalanine balance, rate of appearance or rate of disappearance were found between patients and controls under fasting conditions. However, the anabolic response to hyperinsulinaemia-hyperaminoacidaemia was reduced by more than 50% in patients compared with controls. The diminished anabolic response was due to reduced suppression of the leg phenylalanine appearance rate, an index of protein breakdown, in HF patients; whereas no group difference was found in the increase in the leg phenylalanine disappearance rate, an index of protein synthesis. The diminished responses of both phenylalanine balance and appearance rate to hyperinsulinaemia-hyperaminoacidaemia were related to greater circulating IL-6 (interleukin-6) levels. Our results suggest that, following a brief period of nutritional deprivation, HF patients demonstrate an impaired muscle protein anabolic response to meal-related stimuli, due to an inability to suppress muscle proteolysis, and that this diminished protein anabolic response correlates with markers of immune activation. The inability to stimulate muscle protein anabolism following periods of nutritional deficiency may contribute to muscle wasting in HF patients.

Effects of aging and gender on muscle mass and regulation of Akt-mTOR-p70s6k related signaling in the F344BN rat model

Sarcopenia is the loss of muscle mass and strength which occurs with aging. Whether the molecular basis of sarcopenia differs with muscle type and across sex is not well understood. Here we examine how aging affects the regulation of protein kinase B (Akt), the mammalian target of rapamycin (mTOR), AMP activated kinase (AMPK), p70 ribosomal S6 kinase (p70s6k), S6 ribosomal protein (rps6) and calcineurin (CaN) in the slow soleus and fast extensor digitorum longus (EDL) muscles of 6- (adult), 30- (aged), and 36-month (very aged) male and 6- (adult), 26- (aged), and 30-month (very aged) female Fischer 344 × Brown Norway (F344BN) rats. In male animals, soleus and EDL muscle to body weight ratios decreased steadily with age while in the females, losses remained unchanged after 26 months. These age-related changes in the degree of muscle atrophy across sex were associated with differences in the regulation of Akt, mTOR, and p70s6k in the slow-twitch soleus and the regulation of AMPK, 4EBP1, p70s6k and rpS6 in the fast-twitch EDL. Irrespective of muscle type, aging in both the genders was associated with increased calcineurin expression. Taken together, these data suggest that indices of protein synthesis and muscle adaptation are regulated differently with aging in different muscle types and sex.

Absence of insulin signalling in skeletal muscle is associated with reduced muscle mass and function: evidence for decreased protein synthesis and not increased degradation

Loss of skeletal muscle mass and function is observed in many insulin-resistant disease states such as diabetes, cancer cachexia, renal failure and ageing although the mechanisms for this remain unclear. We hypothesised that impaired insulin signalling results in reduced muscle mass and function and that this decrease in muscle mass and function is due to both increased production of atrogenes and aberrant reactive oxygen species (ROS) generation. Maximum tetanic force of the extensor digitorum longus of muscle insulin receptor knockout (MIRKO) and lox/lox control mice was measured in situ. Muscles were removed for the measurement of mass, histological examination and ROS production. Activation of insulin signalling pathways, markers of muscle atrophy and indices of protein synthesis were determined in a separate group of MIRKO and lox/lox mice 15 min following treatment with insulin. Muscles from MIRKO mice had 36% lower maximum tetanic force generation compared with muscles of lox/lox mice. Muscle fibres of MIRKO mice were significantly smaller than those of lox/lox mice with no apparent structural abnormalities. Muscles from MIRKO mice demonstrated absent phosphorylation of AKT in response to exogenous insulin along with a failure to phosphorylate ribosomal S6 compared with lox/lox mice. Atrogin-1 and MuRF1 relative mRNA expression in muscles from MIRKO mice were decreased compared with muscles from lox/lox mice following insulin treatment. There were no differences in markers of reactive oxygen species damage between muscles from MIRKO mice and lox/lox mice. These data support the hypothesis that the absence of insulin signalling contributes to reduced muscle mass and function though decreased protein synthesis rather than proteasomal atrophic pathways.

The Effects of Growth Hormone and/or Testosterone on Whole Body Protein Kinetics and Skeletal Muscle Gene Expression in Healthy Elderly Men: A Randomized Controlled Trial

Alterations of protein turnover may contribute to the progressive decline of muscle mass with aging. Our objective was to examine the effects of near-physiological recombinant human GH and/or testosterone (T) administration to older men on whole body protein kinetics and muscle gene expression. A 6-month randomized, double-blind, placebo-controlled trial in 21 healthy elderly men aged 65-75 yr, was performed. Participants were randomized to receive placebo GH and placebo T, rhGH and placebo T (GH), T and placebo GH (T), or rhGH and T (GHT). The leucine rate of appearance (index of proteolysis), nonoxidative leucine disposal rate (an index of protein synthesis), and leucine oxidation rate were measured with an infusion of l-[1-(13)C] leucine. Muscle biopsies for the measurement of gene expression were performed. Body composition and aerobic capacity (maximal oxygen capacity) were measured. Serum IGF-I levels increased significantly with GH and GHT (P < 0.001) compared with placebo. T increased significantly only in the T group (P = 0.028). Leucine rate of appearance and nonoxidative leucine disposal rate increased with GH (P = 0.015, P = 0.019) and GHT (P = 0.017, P = 0.02), but leucine oxidation did not change significantly in any treatment group. Midthigh muscle mass and maximal oxygen capacity increased (P < 0.04) with GHT only. Expression of muscle function genes did not change significantly, but the within-group comparisons revealed a significant increase of androgen receptor expression in the GHT group (P = 0.001). This study showed that 6-month treatment with low-dose GH alone or with T in healthy elderly men produces comparable increments in whole body protein turnover and protein synthesis.

Calorie restriction accelerates the catabolism of lean body mass during 2 wk of bed rest

Background: Muscle inactivity and low energy intake commonly occur in persons with acute or chronic disease, in astronauts during space flight, and during aging.Objective: We used a crossover design to investigate the effects of the interactions of inactivity and calorie restriction on whole-body composition and protein kinetic regulation in 9 healthy volunteers.Design: Lean body mass (LBM) was measured by using dual-energy X-ray absorptionmetry before and at the end of 14-d periods of bed rest (B) and controlled ambulation (A) in patients receiving eucaloric (E) or hypocaloric (H) (≈80% of total energy expenditure) diets. Whole-body leucine kinetics were determined at the end of the 4 study periods by using a standard stable-isotope technique in the postabsorptive state and during a 3-h infusion of a 0.13 g · kg LBM−1 · h−1 amino acid mixture.Results: In the postabsorptive state, we found a significant (P = 0.04) bed rest × hypocaloric diet interaction for the rate of leucine oxidation, an index of net protein catabolism (A+E: 0.23 ± 0.01; B+E: 25 ± 0.01; A+H: 0.23 ± 0.01; B+H: 0.28 ± 0.01 μmol · min−1 · kg LBM−1). Bed rest significantly (P < 0.01) decreased amino acid–mediated stimulation of nonoxidative leucine disappearance, an index of protein synthesis (A+E: 35 ± 2%; B+E: 30 ± 2%; A+H: 41 ± 3%; B+H: 32 ± 2%). B+H decreased LBM by 1.10 ± 0.1 kg, which is significantly (P < 0.01) greater than the decrease seen with A+E, A+H, or B+E.Conclusion: Calorie restriction enhanced the catabolic response to inactivity by combining greater protein catabolism in the postabsorptive state with an impaired postprandial anabolic utilization of free amino acids.

Impact of Acute and Chronic Low-Dose Glucocorticoids on Protein Metabolism

High-dose glucocorticoids cause acute protein loss by increasing protein breakdown and oxidation. Whether lower glucocorticoid doses, typical of therapeutic use, induce sustained catabolism has not been studied. Our objective was to assess the effect of acute and chronic therapeutic glucocorticoid doses on protein metabolism. We conducted an open longitudinal and a cross-sectional study at a clinical research facility. Ten healthy subjects were studied before and after a short course of prednisolone (5 and 10 mg/d sequentially for 7 d each). Twelve subjects with inactive polymyalgia rheumatica receiving chronic (>12 months) prednisone (mean = 5.0 +/- 0.8 mg/d) were compared with 12 age- and gender-matched normal subjects. Whole-body protein metabolism was assessed using a 3-h primed constant infusion of 1-[(13)C]leucine, from which rates of leucine appearance (leucine Ra, an index of protein breakdown), leucine oxidation (Lox, index of protein oxidation) and leucine incorporation into protein (LIP, index of protein synthesis) were estimated. Prednisolone induced an acute significant increase in Lox (P = 0.008) and a fall in LIP (P = 0.08) but did not affect leucine Ra. There was no significant difference between the effects of the 5- and 10-mg prednisolone doses on leucine metabolism. In subjects receiving chronic prednisone therapy, leucine Ra, Lox, and LIP were not significantly different from normal subjects. Glucocorticoids stimulate protein oxidation after acute but not chronic administration. This time-related change suggests that glucocorticoid-induced stimulation of protein oxidation does not persist but that a metabolic adaptation occurs to limit protein loss.

Protein Metabolism in Acromegaly: Differential Effects of Short- and Long-Term Treatment

GH acutely increases body protein by stimulating protein synthesis and reducing protein oxidation. The objective of the study was to determine whether these changes in protein metabolism are sustained in long-term GH excess and reversed by correction. We conducted a cross-sectional study in 16 acromegalic and 18 normal subjects and a longitudinal study in which acromegalic subjects were studied before and after short-term (n=8) or long-term (n=10) treatment. The study was conducted at a clinical research center. Whole-body rates of leucine appearance (leucine Ra; an index of protein breakdown), leucine oxidation, and nonoxidative leucine disposal (NOLD; an index of protein synthesis) estimated using infusion of 1-[13C] leucine were measured. Leucine Ra and NOLD were greater (P<0.01) in acromegalic compared with normal subjects, whereas leucine oxidation did not differ. Leucine oxidation increased significantly (P<0.05) after short-term treatment but returned to baseline after long-term treatment. Both leucine Ra and NOLD decreased significantly (P<0.05) after short- and long-term treatment. Adjustment for body composition did not affect results. In acromegalic subjects, protein breakdown and synthesis are increased, whereas protein oxidation does not differ from normal subjects. Protein oxidation increases transiently, whereas protein breakdown and synthesis are stably reduced after treatment. Because protein oxidation represents irreversible loss, we conclude that the normal state of protein oxidation found in acromegaly and after long-term treatment represents metabolic adaptation, which maintains protein mass at a steady state after stable changes in GH status.

Elevated glucose activates protein synthesis in cultured cardiac myocytes

Diabetes mellitus results in chronic hyperglycemia, a serious metabolic disorder associated with a markedly increased risk of cardiovascular disease. However, the effects of high glucose (HG) on cardiac myocyte growth have not been fully clarified. In this study, the effect of glucose on cardiac myocyte growth was examined using leucine incorporation as an index of protein synthesis. High glucose (HG, 25 mmol/L) increased leucine incorporation (167% ± 0.2% over normal glucose, n = 4, P < .01) compared with a physiological glucose concentration (5.5 mmol/L, normal glucose). The HG-induced increase in leucine incorporation was time- and dose-dependent and was not due to osmotic changes because 25 mmol/L mannitol did not change leucine incorporation. High glucose also significantly reduced elongation factor 2 phosphorylation, an effect known to result in increased protein synthesis at the elongation step. Western blot analysis showed that HG-activated protein kinase B (PKB), also called Akt (PKB/Akt), at 18 hours. High glucose–induced leucine incorporation was attenuated with phosphatidylinositol 3-kinase (PI3K) inhibition using wortmannin and LY294002 and by rapamycin, a mammalian target of rapamycin (mTOR) inhibitor, 72%, 64%, and 65% ( P < .05), respectively. High glucose also activated extracellular signal–regulated kinase 1/2 activity with peak stimulation at 5 minutes. In addition, PD98059, an inhibitor of mitogen-activated protein kinase kinase, attenuated HG-induced leucine incorporation. These data show for the first time that elevated glucose increases protein synthesis in cardiac myocytes. The increase appears to be mediated by activation of PI3K-PKB/Akt and/or PI3K-mTOR as well as extracellular signal–regulated kinase 1/2. These results provide new evidence for a direct effect of glucose independent of insulin on cardiac myocyte growth.

Whole body leucine flux in HIV-infected patients treated with or without protease inhibitors

The present study was carried out to assess the effects of protease inhibitor (PI) therapy on basal whole body protein metabolism and its response to acute amino acid-glucose infusion in 14 human immunodeficiency virus (HIV)-infected patients. Patients treated with PIs (PI+, 7 patients) or without PIs (PI-, 7 patients) were studied after an overnight fast during a 180-min basal period followed by a 140-min period of amino acid-glucose infusion. Protein metabolism was investigated by a primed constant infusion of l-[1-(13)C]leucine. Dual-energy X-ray absorptiometry for determination of fat-free mass (FFM) and body fat mass measured body composition. In the postabsorptive state, whole body leucine balance was 2.5 times (P < 0.05) less negative in the PI+ than in the PI- group. In HIV-infected patients treated with PIs, the oxidative leucine disposal during an acute amino acid-glucose infusion was lower (0.58 +/- 0.09 vs. 0.81 +/- 0.07 micromol x kg FFM(-1) x min(-1) using plasma [(13)C]leucine enrichment, P = 0.06; or 0.70 +/- 0.10 vs. 0.99 +/- 0.08 micromol x kg FFM(-1) x min(-1) using plasma [(13)C]ketoisocaproic acid enrichment, P = 0.04 in PI+ and PI- groups, respectively) than in patients treated without PIs. Consequently, whole body nonoxidative leucine disposal (an index of protein synthesis) and leucine balance (0.50 +/- 0.10 vs. 0.18 +/- 0.06 micromol x kg FFM x (-1) x min(-1) in PI+ and PI- groups respectively, P < 0.05) were significantly improved during amino acid-glucose infusion in patients treated with PIs. However, whereas the response of whole body protein anabolism to an amino acid-glucose infusion was increased in HIV-infected patients treated with PIs, any improvement in lean body mass was detected.

Parasympathetic nerve-evoked protein synthesis, mitotic activity and salivary secretion in the rat parotid gland and the dependence on NO-generation

Incorporation of radiolabelled leucine and thymidine into trichloroacetic acid-insoluble material of the parotid gland was used as indices of protein synthesis and mitotic activity, respectively, following electrical stimulation of the parasympathetic auriculo-temporal nerve for 30 min in pentobarbitone-anaesthetized rats under adrenoceptor blockade (phentolamine and propranolol, 2mg/kg intravenous of each) in the absence or presence of atropine (2mg/kg intravenous) and without or with nitric oxide synthase inhibitors. In atropinized rats, the parasympathetic non-adrenergic, non-cholinergic (NANC) nerve-evoked mean increases in protein synthesis at a frequency of 10 Hz (142%) and 40 Hz (200%) were not affected in a statistically significant way (124 and 275%, respectively) by the neuronal type NO-synthase inhibitor N(w)propyl-l-arginine (N-PLA) (30 mg/kg intravenous). Neither were the increase (175%) in protein synthesis at 10 Hz in non-atropinized animals affected by N-PLA (180%). The increase (65%) in mitotic activity, 19 h after the end of stimulation at 40 Hz, in the presence of atropine, was not affected by N-PLA (55%). Neither were the increase (95%) in gland content of amylase at this point of observation statistically significant affected by N-PLA (144%). The secretion of fluid and output of amylase from the parotid gland upon nerve stimulation was not affected by N-PLA. When examining the non-selective NO-synthase inhibitor l-NAME (30 mg/kg intravenous) in atropinized rats subjected to stimulation at 10 Hz, neither the increase in protein synthesis nor the evoked fluid response or amylase outputs were affected. Hence, in contrast to an NO-dependent sympathetic-induced protein synthesis and mitosis in the parotid gland, involving the activity of the neuronal type NO-synthase, no support for a parasympathetic-induced protein synthesis (and gain in gland amylase) and mitosis, depending on NO-generation, was found. Likewise, the present findings provide no evidence for a role of NO in the parasym pathetic nerve-evoked fluid secretion and amylase output.

Mitogen-activated Protein Kinases and Mitogen-activated Protein Kinase Phosphatases Mediate the Inhibitory Effects of All-trans Retinoic Acid on the Hypertrophic Growth of Cardiomyocytes

All-trans retinoic acid (RA) has been implicated in mediation of cardiac growth inhibition in neonatal cardiomyocytes. However, the associated signaling mechanisms remain unclear. Utilizing neonatal cardiomyocytes, we demonstrated that RA suppressed the hypertrophic features induced by cyclic stretch or angiotensin II (Ang II). Cyclic stretch- or Ang II-induced activation of extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (MAP kinase) was dose- and time-dependently inhibited by RA. Significant inhibition was observed by 5 microm RA, from 8 to 24 h of pretreatment. This inhibitory effect was not mediated at the level of mitogen-activated protein kinase kinases (MKKs), because RA had no effect on stretch- or Ang II-induced phosphorylation of MEK1/2, MKK4, and MKK3/6. However, the phosphatase inhibitor vanadate reversed the inhibitory effect of RA on MAP kinases and protein synthesis. RA up-regulated the expression level of MAP kinase phosphatase-1 (MKP-1) and MKP-2, and the time course was correlated with the inhibitory effect of RA on activation of MAP kinases. Overexpression of wild-type MKP-1 inhibited the phosphorylation of JNK and p38 in cardiomyocytes. These data indicated that MKPs were involved in the inhibitory effect of RA on MAP kinases. Using specific RAR and RXR antagonists, we demonstrated that both RARs and RXRs were involved in regulating stretch- or Ang II-induced activation of MAP kinases. Our findings provide the first evidence that the anti-hypertrophic effect of RA is mediated by up-regulation of MKPs and inhibition of MAP kinase signaling pathways.

Nitric oxide-dependent protein synthesis in parotid and submandibular glands of anaesthetized rats upon sympathetic stimulation or isoprenaline administration.

In anaesthetized female rats, the beta-adrenoceptor agonist isoprenaline was intravenously infused (20 microg kg(-1) min(-1)) for 30 min or the ascending cervical sympathetic nerve trunk was intermittently stimulated (50 Hz, 1 s every tenth second) on one side for 30 min. The incorporation of [3H]leucine into trichloroacetic acid (TCA)-insoluble material was used as an index of protein synthesis. In response to isoprenaline, the [3H]leucine incorporation increased by 79% in the parotid glands and by 82% in the submandibular glands. The neuronal type NO-synthase inhibitor N-PLA, reduced (P < 0.001) this response to 26% and 20%, respectively. Sympathetic stimulation under alpha-adrenoceptor blockade increased the [3H]leucine incorporation by 192% in the parotid glands and by 35% in the submandibular glands. N-PLA reduced the corresponding percentage figures to 86% (P < 0.01) and 8% (P < 0.05). When tested in the parotid glands, the non-selective NO-synthase inhibitor L-NAME reduced (P < 0.01) the nerve-evoked response to 91%. The increase in [3H]leucine incorporation in response to sympathetic stimulation under beta-adrenoceptor blockade was not affected by N-PLA in the parotid (139% versus 144%) and submandibular glands (39% versus 34%). In non-stimulated glands, the [3H]leucine incorporation was not influenced by the NO-synthase inhibitors. In conclusion, beta-adrenoceptor mediated salivary gland protein synthesis is largely dependent on NO generation by neuronal type NO-synthase, most likely of parenchymal origin.

Involvement of Protein Kinase C Activation in L-Leucine-Induced Stimulation of Protein Synthesis in L6 Myotubes

Effects of leucine and related compounds on protein synthesis were studied in L6 myotubes. The incorporation of [(3)H]tyrosine into cellular protein was measured as an index of protein synthesis. In leucine-depleted L6 myotubes, leucine and its keto acid, alpha-ketoisocaproic acid (KIC), stimulated protein synthesis, while D-leucine did not. Mepacrine, an inhibitor of both phospholipases A(2) and C, canceled stimulatory actions of L-leucine and KIC on protein synthesis. Neither indomethacin, an inhibitor of cyclooxygenase, nor caffeic acid, an inhibitor of lipoxygenase, diminished their stimulatory actions, suggesting no involvement of arachidonic acid metabolism. Conversely, 1-O-hexadecyl-2-O-methylglycerol, an inhibitor of proteinkinase C, significantly canceled the stimulatory actions of L-leucine and KIC on protein synthesis, suggesting an involvement of phosphatidylinositol degradation and activation of protein kinase C. L-Leucine caused a rapid activation of protein kinase C in both cytosol and membrane fractions of the cells. These results strongly suggest that both L-leucine and KIC stimulate protein synthesis in L6 myotubes through activation of phospholipase C and protein kinase C.

Can Glutamine and Growth Hormone Promote Protein Anabolism in Children with Cystic Fibrosis?

To determine whether recombinant human growth hormone (rhGH), glutamine (GLN) or a combination of both agents can enhance protein synthesis in cystic fibrosis (CF) patients, six 9.6 ± 0.5-year-old prepubertal children (4 M, 2 F) with CF and stable lung disease with undernutrition (weight/height <50th percentile) or delayed growth (height <5th percentile) received stable isotope infusions, in the postabsorptive state and on 4 separate study days: (a) at baseline, and after a 4-week treatment with either, (b) oral GLN (0.7 g/kg/day), (c) rhGH (0.3 mg/kg/week, SC), or (d) both GLN and rhGH. Four-hour infusions of ¹³C-leucine were used to assess leucine appearance rate (Ra, an index of protein breakdown), oxidation (Ox), and non-oxidative leucine disposal (NOLD, an index of protein synthesis). Results are expressed as changes (%) from baseline:We conclude that in children with CF: (1) due to high inter-subject variability, oral glutamine does not significantly enhance protein gain; (2) rhGH has significant anabolic effects which are mediated by stimulation of protein synthesis, and (3) glutamine does not enhance the effect of rhGH.