Modulation of muscle protein synthesis by insulin is maintained during neonatal endotoxemia

Sepsis promotes insulin resistance and reduces protein synthesis in skeletal muscle of adults. The effect of sepsis on insulin-stimulated muscle protein synthesis has not been determined in neonates, a highly anabolic population that is uniquely sensitive to insulin. Overnight fasted neonatal pigs were infused for 8 h with endotoxin [lipopolysaccharide (LPS), 0 and 10 mug.kg(-1).h(-1)]. Glucose and amino acids were maintained at fasting levels, insulin was clamped at either fasting or fed (2 or 10 muU/ml) levels, and fractional protein synthesis rates were determined at the end of the infusion. LPS infusion induced a septic-like state, as indicated by a sustained elevation in body temperature, heart rate, and cortisol. At fasting insulin levels, LPS reduced fractional protein synthesis rates in gastrocnemius muscle (-26%) but had no effect on the masseter and heart. By contrast, LPS stimulated liver protein synthesis (+28%). Increasing insulin to fed levels accelerated protein synthesis rates in gastrocnemius (controls by +38%, LPS by +60%), masseter (controls by +50%, LPS by +43%), heart (controls by +34%, LPS by +40%), and diaphragm (controls by +54%, LPS by +29%), and the response to insulin was similar in LPS and controls. Insulin did not alter protein synthesis in liver, kidney, or jejunum in either group. These findings suggest that acute endotoxemia lowers basal fasting muscle protein synthesis in neonates but does not alter the response of protein synthesis to insulin.     

Regulation of protein synthesis in lactating rat mammary tissue by cell volume

The effect of changing cell volume on rat mammary protein synthesis has been examined. Cell swelling, induced by a hyposmotic challenge, markedly increased the incorporation of radiolabelled amino acids (leucine and methionine) into trichloroacetic acid (TCA)-precipitable material: reducing the osmolality by 47% increased leucine and methionine incorporation into mammary protein by 147 and 126% respectively. Conversely, cell shrinking, induced by a hyperosmotic shock, almost abolished the incorporation of radiolabelled amino acids into mammary protein: increasing the osmolality by 70% reduced leucine and methionine incorporation into mammary protein by 86 and 93% respectively. The effects of cell swelling and shrinking were fully reversible. Volume-sensitive mammary tissue protein synthesis was dependent upon the extent of the osmotic challenge. Isosmotic swelling of mammary tissue, using a buffer containing urea (160 mM), increased the incorporation of radiolabelled leucine into TCA-precipitable material by 106%. Swelling-induced mammary protein synthesis was dependent upon calcium: removing extracellular calcium together with the addition of EGTA markedly reduced volume-activated protein synthesis. Cell swelling-induced protein synthesis was inhibited by the Ca(2+) ATPase blocker thapsigargin suggesting that volume-sensitive protein synthesis is dependent upon luminal calcium.     

Macromolecular synthesis in E. coli, isolated mitochondria and L5178Y cells in the presence of hydroxyurea or formamidoxime

The effects of formamidoxime and hydroxyurea over a 10⁵ concentration range were studied on macromolecular synthesis in E. coli, L5178Y mouse leukemic cells, isolated rat liver mitochondria and isolated rat cerebral cortex mitochondria. In E. coli 2 mg per ml of formamidoxime and hydroxyurea inhibited, respectively, RNA synthesis by 20% and 17%, DNA synthesis by 91% and 96%, protein synthesis by 54% and 60% and lipopolysaccharide synthesis by 65% and 48%. In L5178Y mouse leukemic cells 2 mg/ml of formamidoxime and hydroxyurea inhibited, respectively, RNA synthesis by 41% and 24%, DNA synthesis by 90% and 97%, protein synthesis by 59% and 44% and glycoprotein synthesis by 83% and 50%. In isolated rat liver mitochondria 2 mg/ml of formamidoxime and hydroxyurea inhibited, respectively, RNA synthesis by 43% and 52%, DNA synthesis by 42% and 56% and protein synthesis by 18% and 30%. Glycoprotein synthesis was not affected. In isolated rat cerebral cortex mitochondria 2 mg/ml of formamidoxime and hydroxyurea inhibited, respectively, RNA synthesis by 50% and 44%, DNA synthesis by 59% and 66% and protein synthesis by 48% and 40%. Glycoprotein synthesis again was not affected. Lower concentrations of the drugs produced less inhibition of macromolecular synthesis in each of the systems.     

The effects of endotoxaemia on protein metabolism in skeletal muscle and liver of fed and fasted rats.

The response of muscle and liver protein metabolism to either a single or three successive daily injections of an endotoxin (Escherichia coli lipopolysaccharide, serotype 0127 B8; 1 mg/ml, 0.3 mg/100 g body wt.) was studied in vivo in the fed rat, and at 24 and 30 h after endotoxin treatment during fasting. In the fed rats there was a catabolic response in muscle, owing to a 60-100% increase in muscle protein degradation rate, and a 52% fall in the synthesis rate. Although there was a 20% decrease in food intake, the decrease in protein synthesis was to some extent independent of this, since rats treated with endotoxin and fasted also showed a lower rate of muscle protein synthesis, which was in excess of the decrease caused by fasting alone. The mechanism of this decreased protein synthesis involved decreased translational activity, since in both fed and fasted rats there was a decreased rate of synthesis per unit of RNA. This occurred despite the fact that insulin concentrations were either maintained or increased, in the fasted rats, to those observed in fed rats. In the liver total protein mass was increased in the fed rats by 16% at 24 h, and the fractional synthesis rate at that time was increased by 35%. In rats fasted after endotoxin treatment the liver protein mass was not decreased as it was in the control fasted rats, and the fractional synthesis rate was increased by 22%. In both cases the increased synthesis rate reflected an elevated hepatic RNA concentration. The extent of this increase in hepatic protein synthesis was sufficient at one point to compensate for the fall in estimated muscle protein synthesis, so that the sum total in the two tissues was maintained.     

Wound tumor virus polypeptide synthesis in productive noncytopathic infection of cultured insect vector cells.

Inoculation of the leafhopper cell line AC-20 with wound tumor virus resulted in a productive noncytopathic infection with no detectable alteration of cellular protein synthesis. Virus-specific polypeptide synthesis, detectable by 8 h postinoculation, increased in a linear fashion, reaching a peak (approximately 10 to 15% of total protein synthesis) by 48 h postinoculation. The rate of viral protein synthesis continued at this level for several days but declined, relative to cellular protein synthesis, as infected cells were passaged. By passage 10, the synthesis of viral polypeptides was reduced to a level approximately 5% of that observed at 48 h postinoculation. Viral protein synthesis was not stimulated by superinfection. Viral antigens and infectious virus persisted in the majority (greater than 90%) of cells in an infected culture even after more than 100 passages. The synthesis of wound tumor virus polypeptides in infected insect vector cells appears to be regulated in a coordinated and selective manner.     

Effect of age and dietary restriction on protein synthesis by isolated kidney cells

The influence of age and food restriction on kidney protein synthesis was studied in Fischer F344 rats. The rate of total protein synthesis by suspensions of kidney cells declined 60% between 4 and 31 months of age. The rate of protein synthesis by kidney cells isolated from 19-month old rats fed a restricted diet (60% of diet consumed by rats fed ad libitum) was 45% higher than the rate of protein synthesis by kidney cells isolated from 19-month old rats fed ad libitum. The excretion of protein in the urine was measured to assess the effect of the age related decline in protein synthesis on kidney function. A dramatic increase in proteinuria was observed with increasing age, and rats fed the restricted diet excreted significantly less protein in the urine than rats fed ad libitum.     

The effect of intermittent changes in tension on protein and collagen synthesis in isolated rabbit muscles.

Isolated intact rabbit muscles were incubated in a medium containing radioactive proline. The rates of synthesis of collagen and total muscle protein after incubation with a constant tension or intermittent mechanical stretching were compared with the rates in vivo. Muscles incubated under a constant tension synthesized protein at 22% of the rate observed in vivo; intermittent mechanical stretching resulted in an increase of 73% in the rate of protein synthesis, to 38% of that found in vivo. Collagen synthesis was affected in the same way as total protein synthesis by both types of incubation, therefore the relative rates of collagen and total protein synthesis were unchanged. ATP concentration in the isolate muscles and the uptake of glucose from the medium were increased by intermittent mechanical stretching. Incubating the muscles with a gas phase containing 5% O2 decreased the rate of protein synthesis, abolished the effect of intermittent mechanical stretching, lowered the concentration of ATP and increased the lactate concentration. The rate of protein synthesis in muscles maintained with a constant or intermittently applied tension was not affected by a previous period of incubation with the other type of stimulus.     

Epinephrine and glucagon counteract inhibition of protein synthesis induced by D-galactosamine in isolated mouse hepatocytes.

10 mM D-galactosamine enhibited protein synthesis (1 h incubation time) by 67% in isolated mouse liver cells. Counteracting uridylate deficiency induced by D-galactosamine by preventive administration of 20 mM uridine did not decrease the extent of protein synthesis inhibition. 20 mM D-galactose reverted the inhibition of protein synthesis by D-galactosamine. 10(-5) M epinephrine and 10(-7) M glucagon decreased the incorporation of D-galactosamine into glycogen to 38% and 26% of the control value, respectively, after a 35 min incubation and reduced the inhibition of protein synthesis by D-galactosamine effectively. Experimental evidence supports the view that aminoglycogen formed after D-galactosamine treatment is responsible for the inhibition of protein synthesis.     

Indomethacin inhibits the insulin-induced increases in RNA and protein synthesis in L6 skeletal muscle myoblasts

Rates of accretion of RNA and protein and rates of protein synthesis were measured in sub-confluent cultures of L6 myoblasts. Insulin (100 microU/ml) stimulated protein synthesis by 15% within 30 min and by 40% at two and six hours. By six hours insulin also increased the accretion of RNA (+15%). The cyclo-oxygenase inhibitor indomethacin did not reduce the basal rate of RNA or protein accretion in L6 cells but reduced the rate of protein synthesis by 16%. When added together with insulin, indomethacin inhibited the hormonally-stimulated rate of protein synthesis and also significantly reduced the accretion of RNA. Indomethacin still reduced the effects of insulin on protein synthesis when added to the cells two hours after the hormone. Synthesis of RNA measured by the incorporation of [3H]-uridine was also stimulated by insulin but was inhibited by indomethacin only when the drug was present throughout the incubation. Inhibition of protein synthesis by cyclo-oxygenase inhibitors may be the result of both a direct action on translational efficiency and an effect on RNA synthesis.     

Indomethacin inhibits the insulin-induced increases in RNA and protein synthesis in L6 skeletal muscle myoblasts

Rates of accretion of RNA and protein and rates of protein synthesis were measured in sub-confluent cultures of L6 myoblasts. Insulin (100 μU/ml) stimulated protein synthesis by 15% within 30 min and by 40% at two and six hours. By six hours insulin also increased the accretion of RNA (+ 15%). The cyclo-oxygenase inhibitor indomethacin did not reduce the basal rate of RNA or protein accretion in L6 cells but reduced the rate of protein synthesis by 16%. When added together with insulin, indomethacin inhibited the hormonally-stimulated rate of protein synthesis and also significantly reduced the accretion of RNA. Indomethacin still reduced the effects of insulin on protein synthesis when added by the incorporation of [³H]-uridine was also stimulated by insulin but was inhibited by indomethacin only when the drug was present throughout the incubation. Inhibition of protein synthesis by cyclo-oxygenase inhibitors may be the result of both a direct action on translational efficiency and an effect on RNA synthesis.     

The effect of insulin and intermittent mechanical stretching on rates of protein synthesis and degradation in isolated rabbit muscle.

Tyrosine balance and protein synthesis were studied during the same incubation in isolated rabbit forelimb muscles. From these measurements, protein degradation was calculated. Isolated muscles were usually in a state of negative amino acid balance, principally as a result of the 75% decrease in protein synthesis. Muscles from rabbits starved for 18 h had lower rates of both protein synthesis and degradation compared with muscles from normally fed rabbits. Intermittent mechanical stretching and the addition of insulin at 100 microunits/ml increased rates of both protein synthesis and degradation. Increases in the rate of protein synthesis were proportionately greater in the muscles from starved animals. In muscles from both fed and starved donors, increases in protein-synthesis rates owing to intermittent stretching and insulin were proportionately greater than the increases in degradation rates. For example, insulin increased the rate of protein synthesis in the muscles from starved donors by 111% and the rate of degradation by 31%. Insulin also increased the rate of protein synthesis when added at a higher concentration (100 munits/ml); at this concentration, however, the rate of protein degradation was not increased. The suppressive effect of insulin on high rates of protein degradation in other skeletal-muscle preparations may reflect a non-physiological action of the hormone.     

Stimulation of epidermal protein synthesis in vivo by topical triamcinolone acetonide.

The rate of epidermal protein synthesis in vivo was determined in the hairless mouse by a method in which a large dose of [3H]phenylalanine (150 mumol/100 g body wt.) is administered via the tail vein. The epidermal free phenylalanine specific radioactivity rapidly rose to a plateau value which by 10 min approached that of plasma, after which it declined. This dose of phenylalanine did not of itself alter protein synthesis rates, since incorporation of co-injected tracer doses of [3H]lysine and [14C]threonine was unaffected. The fractional rate of protein synthesis obtained for epidermis was 61.6%/day, whereas values for liver and gastrocnemius muscle in the same group of mice were 44%/day and 4.8%/day respectively. When expressed on the basis of RNA content, the value for epidermis (18.6 mg of protein/day per mg of RNA) was approx. 3-fold higher than those for liver and gastrocnemius muscle. Topical administration of 0.1% triamcinolone acetonide increased the epidermal fractional protein synthesis rate by 33% after 1 day and by 69% after 7 days, compared with vehicle-treated controls. These effects were entirely accounted for by the increase in protein synthesis rates per mg of RNA. RNA/protein ratios were unaffected by this treatment.     

Analysis of effect of age on synthesis of specific proteins by hepatocytes

The effect of age on the synthesis of specific proteins by hepatocytes was studied in Fischer F344 rats using two-dimensional polyacrylamide gel electrophoresis. Almost all proteins synthesized by hepatocytes from young rats were synthesized by hepatocytes isolated from old rats. Of over 500 proteins visually compared by two-dimensional polyacrylamide gel electrophoresis, only 11 proteins were observed to disappear and/or appear consistently with increasing age. The rates of synthesis of 36 randomly chosen proteins were quantified. Interestingly, the synthesis of 35 of the 36 proteins decreased between 5 and 30 months of age. The decrease in protein synthesis varied (15% to 70%) from one protein to another; i.e., a heterogeneity was observed in the age-related decrease in the synthesis of proteins. The age-related decrease in protein synthesis was statistically significant for 53% of the proteins studied. The total decrease in the rate of synthesis of all 36 proteins studied was 40% between 5 and 30 months of age, which is essentially the same as the decrease in total protein synthesis by suspension of hepatocytes isolated from 5- and 30-month-old rats. The results of this study demonstrate that the mechanism underlaying aging is different from development, which is characterized by a major change in the species of proteins synthesized by a cell.     

Depression of protein synthesis during diapause in embryos of the annual killifish Austrofundulus limnaeus

Rates of protein synthesis are substantially depressed in diapause II embryos of Austrofundulus limnaeus. Inhibition of oxygen consumption and heat dissipation with cycloheximide indicates that 36% of the adenosine triphosphate (ATP) turnover in prediapausing embryos (8 d postfertilization [dpf]) is caused by protein synthesis; the contribution of protein synthesis to ATP turnover in diapause II embryos is negligible. In agreement with the metabolic data, incorporation of amino acids (radiolabeled via (14)CO(2)) into perchloric acid-precipitable protein decreases by over 93% in diapause II embryos compared with embryos at 8 dpf. This result represents a 36% reduction in energy demand because of depression of protein synthesis during diapause. Adjusting for changes in the specific radioactivity of the free amino acid pool at the whole-embryo level yields rates of protein synthesis that are artifactually high and not supportable by the observed rates of oxygen consumption and heat dissipation during diapause. This result indicates a regionalized distribution of labeled amino acids likely dictated by a pattern of anterior to posterior cell cycle arrest. AMP/ATP ratios are strongly correlated with the decrease in rates of protein synthesis, which suggests a role for adenosine monophosphate (AMP) in the control of anabolic processes. The major depression of protein synthesis during diapause II affords a considerable reduction in energy demand and extends the duration of dormancy attainable in these embryos.     

Effect of testosterone on muscle mass and muscle protein synthesis

We have studied the effect of a pharmacological dose of testosterone enanthate (3 mg.kg-1.wk-1 for 12 wk) on muscle mass and total-body potassium and on whole-body and muscle protein synthesis in normal male subjects. Muscle mass estimated by creatinine excretion increased in all nine subjects (20% mean increase, P less than 0.02); total body potassium mass estimated by 40K counting increased in all subjects (12% mean increase, P less than 0.0001). In four subjects, a primed continuous infusion protocol with L-[1-13C]leucine was used to determine whole-body leucine flux and oxidation. Whole-body protein synthesis was estimated from nonoxidative flux. Muscle protein synthesis rate was determined by measuring [13C]leucine incorporation into muscle samples obtained by needle biopsy. Testosterone increased muscle protein synthesis in all subjects (27% mean increase, P less than 0.05). Leucine oxidation decreased slightly (17% mean decrease, P less than 0.01), but whole-body protein synthesis did not change significantly. Muscle morphometry showed no significant increase in muscle fiber diameter. These studies suggest that testosterone increases muscle mass by increasing muscle protein synthesis.     

Stimulation of protein synthesis by both insulin and amino acids is unique to skeletal muscle in neonatal pigs

In neonatal pigs, the feeding-induced stimulation of protein synthesis in skeletal muscle, but not liver, can be reproduced by insulin infusion when essential amino acids and glucose are maintained at fasting levels. In the present study, 7- and 26-day-old pigs were studied during 1) fasting, 2) hyperinsulinemic-euglycemic-euaminoacidemic clamps, 3) euinsulinemic-euglycemic-hyperaminoacidemic clamps, and 4) hyperinsulinemic-euglycemic-hyperaminoacidemic clamps. Amino acids were clamped using a new amino acid mixture enriched in nonessential amino acids. Tissue protein synthesis was measured using a flooding dose of L-[4-(3)H]phenylalanine. In 7-day-old pigs, insulin infusion alone increased protein synthesis in various skeletal muscles (from +35 to +64%), with equivalent contribution of myofibrillar and sarcoplasmic proteins, as well as cardiac muscle (+50%), skin (+34%), and spleen (+26%). Amino acid infusion alone increased protein synthesis in skeletal muscles (from +28 to +50%), also with equivalent contribution of myofibrillar and sarcoplasmic proteins, as well as liver (+27%), pancreas (+28%), and kidney (+10%). An elevation of both insulin and amino acids did not have an additive effect. Similar qualitative results were obtained in 26-day-old pigs, but the magnitude of the stimulation of protein synthesis by insulin and/or amino acids was lower. The results suggest that, in the neonate, the stimulation of protein synthesis by feeding is mediated by either amino acids or insulin in most tissues; however, the feeding-induced stimulation of protein synthesis in skeletal muscle is uniquely regulated by both insulin and amino acids.     

Effects of amino acids, ammonia and leupeptin on protein synthesis and degradation in isolated rat hepatocytes.

Protein synthesis in isolated rat hepatocytes, as measured by the incorporation of [14C]-valine at constant specific radioactivity, proceeded at a rate of 0.3-0.5%/h in an unsupplemented medium, i.e. only about one-tenth the rate of protein degradation (4%/h). Leupeptin, which inhibits lysosomal protein degradation (previously found to be 75% of the total degradation in hepatocytes), had no effect on protein synthesis, showing that endogenous protein degradation supplied amino acids in excess of the substrate requirements for protein synthesis. The inhibition of protein synthesis by NH4Cl (another inhibitor of lysosomal protein degradation) as well as the stimulation by a physiological amino acid mixture must therefore represent indirect effects, either on general energy metabolism, or on unknown regulatory processes.     

Control of Neuronal Size Homeostasis by Trophic Factor–mediated Coupling of Protein Degradation to Protein Synthesis

We demonstrate that NGF couples the rate of degradation of long-lived proteins in sympathetic neurons to the rate of protein synthesis. Inhibiting protein synthesis rate by a specific percentage caused an almost equivalent percentage reduction in the degradation rate of long-lived proteins, indicating nearly 1:1 coupling between the two processes. The rate of degradation of short-lived proteins was unaffected by suppressing protein synthesis. Included in the pool of proteins that had increased half-lives when protein synthesis was inhibited were actin and tubulin. Both of these proteins, which had half-lives of several days, exhibited no degradation over a 3-d period when protein synthesis was completely suppressed. The half-lives of seven other long-lived proteins were quantified and found to increase by 84–225% when protein synthesis was completely blocked.Degradation–synthesis coupling protected cells from protein loss during periods of decreased synthesis. The rate of protein synthesis greatly decreased and coupling between degradation and synthesis was lost after removal of NGF. Uncoupling resulted in net loss of cellular protein and somatic atrophy. We propose that coupling the rate of protein degradation to that of protein synthesis is a fundamental mechanism by which neurotrophic factors maintain homeostatic control of neuronal size and perhaps growth.     

Estimation of growth potential by measurement of tissue protein synthetic rates in feeding and fasting rainbow trout, Salmo gairdnerii Richardson

Protein synthesis in liver, gill and muscle tissue was measured in vivo by constant infusion of ¹⁴C-tyrosine in fed and fasted freshwater rainbow trout, Salmo gairdnerii , at 12° C. Synthesis rates (percentage of tissue protein synthesized per day) were 15-17% in liver, 4–5% in gill and 0.38% in muscle of fed fish. Liver and gill synthesis rate showed no significant change in fish that had been without food for 15 days, whereas muscle protein synthesis fell to 0.09%. The greater susceptability of muscle protein synthesis to fasting, possibly results from the greater proportion of synthesis retained as growth in this tissue. Growth rates indicate little change in protein turnover in the muscle but increased protein degradation with fasting. The difference between fed and fasted synthesis rates in muscle may be used as a measurement of potential growth rate for a particular species.     

Protein synthesis by synaptosomes from rat brain. Contribution by the intraterminal mitochondria

(1) The characteristics of protein synthesis in microsomal and synaptosomal fractions from rat brain were examined. A high sensitivity to ribonuclease and to cycloheximide, and the need for the presence of pH5 enzymes distinguished protein synthesis in microsomal fractions from protein synthesis in synaptosomes. (2) Under various conditions of incubation synaptosomal fractions prepared in sucrose showed limited protein synthesis compared with synaptosomal fractions prepared by using Ficoll. Such discrepancies could not be attributed to: (i) animal age, (ii) the metabolic state of the synaptosomal fraction, (iii) the absence of bivalent cations in the incubation medium or (iv) the temperature. (3) Protein synthesis in synaptosomal fractions was inhibited 50-65% by cycloheximide, 38-50% by chloramphenicol, 95% by puromycin, 70% by azide and 40% by deoxyglucose; ribonuclease had only a negligible inhibitory effect. (4) As a first approximation to the localization of the protein-synthetic machinery present in the synaptosomal fraction, the distribution of enzymes and radioactivity in subfractions of prelabelled synaptosomes was determined after osmotic shock with water. Approximately 60% of the total protein synthesis in the synaptosomal fraction occurred in the intraterminal mitochondria. (5) Protein synthesis in the intraterminal mitochondria did not show any fundamental difference from synthesis in somatic mitochondria, with respect to inhibition by cycloheximide and chloramphenicol. (6) It was concluded that if extramitochondrial protein synthesis occurs in synaptosomes, it must be very low.