Evidence for regulation of protein synthesis at the translation level in response to dietary alterations.

Rats were maintained for several days on a protein-free diet which, approximately 14 hr before decapitation, was changed to a protein-rich diet. Microsomal subfractions of liver were obtained by sucrose density gradient centrifugation. The sedimentation patterns of the microsomal subfractions and of the polysomes were found to be unchanged after the alteration of the diet whereas the ability to incorporate amino acid was markedly enhanced. No significant differences in the RNA/protein ratio of the microsomal subfractions were observed. The changes in amino acid incorporation which were unrelated to concurrent changes in the amount of polysomes are discussed.

Effect of protein diet on age-dependent methylation of liver nuclear components of mice administered N-nitrosodimethylamine.

Protein diets deficient or enriched in DL-methionine were studied for the effect of methylation of nuclear components in subadult and adult outbred albino male mice given N-nitrosodimethylamine. After the mice were fed diets for 6 days, they were given ip injections of [14C]N-nitrosodimethylamine (5 mg/kg body wt) and killed 45 minutes later. Liver nuclei were isolated; incorporation of 14C into nuclear proteins and methylation of DNA and RNA were determined. In the subadult and adult animals the nuclear protein content was diminished by shortage of a single essential amino acid. Incorporation of 14C into the total protein was reduced. The reduction amounted to 55% in the subadult and 23% in the adult animals. Neither an age-dependent nor a diet-dependent change in the specific methylation of DNA and RNA was observed. Analysis of methylation of DNA and RNA was observed. Analysis of methylated purine bases showed an age-related rise in O6-methylguanine in the adult as compared with the subadult mice.

Protein synthesis assessed by ribosome analysis in human papillary muscle in relation to oxidative capacity: a comparison with skeletal muscle.

Protein synthesis as assessed by the concentration and size distribution of ribosomes was determined together with citrate synthase activity in papillary muscles obtained at open heart surgery from patients with mitral valve disease. The results were compared with corresponding data from the quadriceps femoris muscle of patients undergoing cholecystectomy. Citrate synthase activity was six times higher in papillary muscle than in skeletal muscle. The total ribosome concentration per mg DNA was similar in the two types of muscle. Compared with skeletal muscle, in papillary muscle polyribosomes constituted a higher proportion of the ribosomes (p less than 0.001), and there was a tendency towards larger polyribosome aggregates. It is proposed that the high concentration of polyribosomes in papillary muscle is related to the high oxidative capacity of that tissue.

Protein synthesis activity in vitro in mouse tissues in relation to methionine-cysteine deficiency and age.

The objective of this study was to determine whether ageing in the mouse is associated with changes in protein synthesis activity and response to dietary methionine-cysteine deficiency. Growing subadult mice (30-day-old) and adult mice (70-day-old) were compared. The amino acid restricted diet contained methionine and cysteine at a level of 10% that of the control diet. After a feeding period of 6 days protein synthesis activity was measured in liver, kidney and lung. In control liver, the level of protein synthesis activity was the same for the two age groups; in kidney and lung, activity was diminished in the adult group. After methionine-cysteine deficiency protein synthesis activity was unchanged in the subadult and diminished in the adult liver. In kidney, both age groups responded with an increase in activity. In lung, the subadult group remained unchanged and activity in the adult group was elevated. The amount of DNA/g wet weight of tissue was higher in the subadult than the adult group throughout. Methionine-cysteine deficiency resulted in an increase in DNA/g tissue wet weight, while the total amount of DNA in liver decreased, but was unchanged in kidney and lung. Total liver activity was decreased less in subadult than adult mice. Total kidney and lung activities were diminished in subadult but not adult mice. The results suggest that the specific protein synthesis activity of the ribosomes in the kidney and lung tends to decrease with age more readily than in liver, and that subadult liver displays less sensitivity towards methionine-cysteine deficiency than does adult liver.

Nuclear proteins and chromatin structure in liver and intestinal epithelial cells of young growing and adult rats.

Nuclei from liver and intestinal epithelial cells of young growing rats (39 days old) and adult rats (98 days old) were isolated. After addition of DNase I, the chromatin was separated by centrifugation (1100 g) into two fractions; the pellet (P) and the supernatant (S). The amount of chromatin released into the S-fraction was the same for the two age groups. The intestinal epithelial cell nuclei underwent self-digestion (in the absence of added DNase I) which was significantly higher in the young rats than in the adults. Subsequent examination using immunotechniques established the presence of non-sarcomeric myosin heavy-chain indicating that active genes were present for that protein. Hybridization of DNA with cDNA specific for myosin heavy-chain revealed that, relative to total DNA, the DNA retained in the P-fraction of both tissues and age groups contained the same amount of hybridizable sequences. In liver, nuclear proteins decreased significantly with age per g wet weight of tissue. In the enterocyte tissue, total DNA and protein increased with age. SDS-polyacrylamide gel or acetic acid-urea gel electrophoresis gave no age-related differences in the pattern of the proteins within each tissue. The results show that both liver nuclear DNA and protein decrease with age per g wet weight but increase per total tissue. In intestinal epithelial cells changes in chromatin structure with age were inherent within the nucleus.

Methionine-cysteine deficiency and alkylation of DNA in liver, kidney and lung of mice administered dimethylnitrosamine.

The effect of methionine-cysteine deficiency on the methylation of DNA purines by dimethylnitrosamine metabolites was studied in subadult and adult mice. In liver, no dietary effect on the specific methylation of 7-methylguanine was observed, while that of 3-methyladenine decreased in the adult animals. The specific methylation of guanine in the 0(6)-position and the ratio of 0(6)-methylguanine to 7-methylguanine increased significantly after methionine-cysteine deficiency. Methylation in kidney decreased in subadult but increased in adult mice. In lung, the amount of 7-methylguanine was significantly elevated after methionine-cysteine deficiency in both the subadult and adult mice. The results demonstrate an increase in the specific methylation in liver of guanine in the 0(6)-position by the methionine-cysteine deficient diet, together with differences in the methylation pattern between organs of the two age groups.

Alpha-ketoglutarate preserves protein synthesis and free glutamine in skeletal muscle after surgery.

Serving as a reproducible human trauma model, patients (n = 21) undergoing elective cholecystectomy received postoperative total parenteral nutrition with (n = 9) or without (n = 12) alpha-ketoglutarate (AKG) supplementation. Skeletal muscle biopsy specimens were taken before surgery and on the third postoperative day. The postoperative decreases in the concentrations of free glutamine and basic amino acids seen in the control group were counteracted in the AKG group (p less than 0.05). Muscle protein synthesis was estimated by ribosome analysis. On the third postoperative day the control group showed a decline in the polyribosome concentration (25.8% +/- 4.5%; p less than 0.001). No significant change was observed in the AKG group. On each postoperative day the nitrogen balance was negative in the control group but not in the AKG group. In the control group the cumulative nitrogen balance amounted to -9.9 +/- 1.8 gm of nitrogen and in the AKG group -2.6 +/- 2.6 gm of nitrogen, which was significantly different (p less than 0.05). Administration of AKG, the carbon skeleton corresponding to glutamine, produced results similar to those seen when glutamine is added to postoperative total parental nutrition. The results suggest that the availability of precursors for glutamine synthesis in skeletal muscle is crucial for the degree of muscle protein catabolism after surgical trauma.

Polyribosome concentration in human skeletal muscle after starvation and parenteral or enteral refeeding.

Posttraumatic and septic states cause a loss of body proteins resulting in a negative nitrogen balance. The major part of the excreted nitrogen is derived from the proteins of skeletal muscle. The loss in proteins is due to a decrease in protein synthesis rather than an increase in protein degradation. Nutritional support may increase protein synthesis, and determination of its activity in skeletal muscle will give information on the utilization of nutrients in catabolic patients. The effect of nutritional support on healthy subjects was studied to achieve a background for future clinical studies. Male volunteers between 20 and 40 years old were refed parenterally or enterally after three days of starvation. Muscle biopsies (50 mg) were analyzed for the size distribution of ribosomes in a sucrose density gradient, and the ribosome concentration was determined per mg of DNA. Changes in the percentage content of polyribosomes preceded those of the total ribosome concentration. The total polyribosome concentration per gram wet weight of skeletal muscle decreased significantly during starvation. After one and two days of refeeding, a significant increase was observed, but the original level of the nonstarved subjects was not reached. The total ribosome concentration increased upon refeeding, but was not significantly different from that of the starved condition. The nitrogen balance was negative during starvation but attained equilibrium after two days of refeeding. Nutrition administered by the parenteral or enteral route were equally effective in restoring protein synthesis.

Size distribution of ribosomes in biopsy specimens of human skeletal muscle during starvation.

The changes in the poly- and monoribosome distribution and in the total ribosome concentration in muscle during short term starvation were investigated. Transcutaneous muscle biopsies of 50 mg wet wt were taken from healthy human subjects, nonstarved and after one, two, and three days of total starvation. The percentage amount of polyribosomes was significantly lower (P less than 0.02) on days 2 and 3 of starvation than on day 0 (nonstarved). No significant sex-dependent differences were observed between the group of five females and six males. Ribosome concentration per g wet wt of muscle tissue was significantly lower on day 3 than on each preceding day (P less than 0.05). The reproducibility of the polyribosome analyses, together with the changes observed, suggest a future application of this method for evaluation of the effects of nutritional support in patients with posttraumatic and septic conditions.

Stress hormone and amino acid infusion in healthy volunteers: short-term effects on protein synthesis and amino acid metabolism in skeletal muscle.

To study the immediate effects of stress hormones and intravenous amino acid support, healthy male volunteers were administered a stress-hormone infusion including epinephrine, cortisol, and glucagon either alone (Triple, n = 8) or combined with a balanced glutamine-free amino acid solution (Triple AA, n = 8) over a period of 6 hours. The amino acid infusion was started 2 hours after the hormone infusion. A third group (AA, n = 8) received the balanced amino acid solution alone. After 6 hours of the stress-hormone infusion, a decrease was observed in skeletal muscle protein synthesis as measured by the size distribution and concentration of ribosomes. The decrease was prevented by an infusion of the balanced amino acid solution. Following the triple-hormone infusion, a decrease was noted in the content of the total free amino acids in both muscle and plasma. After including amino acids in the infusion solution, the significant decrease in muscle glutamine caused by the triple hormones was not seen. Plasma cortisol, insulin, and glucose increased in response to the triple-hormone infusion alone or in combination with amino acids. In summary, the results show that the signs of muscle protein catabolism elicited by administration of stress hormones can be attenuated by simultaneous administration of a conventional amino acid solution, although it does not contain glutamine.

Glutamine and alpha-ketoglutarate prevent the decrease in muscle free glutamine concentration and influence protein synthesis after total hip replacement.

After surgical trauma, protein synthesis, as well as the concentration of free glutamine in muscle, decreases. Total parenteral nutrition (TPN) alone does not prevent the decrease of glutamine in muscle, but TPN supplemented with glutamine or its precursor, alpha-ketoglutarate, maintains amino acid concentration in muscle and preserves protein synthesis. The aim of this study was to characterize a human trauma model using patients undergoing total hip replacement, and furthermore to investigate whether glutamine or alpha-ketoglutarate alone without TPN can prevent the postoperative decrease in muscle free glutamine. Metabolically healthy patients undergoing total hip replacement were randomized into three groups. The control group (n = 13) received glucose 2 g/kg body weight (BW) during surgery and the first 24 postoperative hours. The glutamine group (n = 10) received glucose 2 g/kg BW and glutamine 0.28 g/kg BW, and the alpha-ketoglutarate group (n = 10) received glucose 2 g/kg BW and alpha-ketoglutarate 0.28 g/kg BW. Muscle biopsies were performed before surgery and 24 hours postoperatively. Free glutamine concentration in muscle decreased from 11.62 +/- 0.67 to 9.80 +/- 0.36 mmol/kg wet weight in the control group (P < .01), whereas it remained unchanged in both the glutamine group and alpha-ketoglutarate group. Protein synthesis, as reflected by the concentration of total ribosomes, decreased significantly in the control group, but not in glutamine and alpha-ketoglutarate groups. Polyribosome concentration decreased significantly in both the control and alpha-ketoglutarate groups. Total hip replacement can be used as a reproducible trauma model, with characteristic changes in the muscle amino acid pattern and protein synthesis 24 hours postoperatively.(ABSTRACT TRUNCATED AT 250 WORDS)

Growth hormone improves muscle protein metabolism and whole body nitrogen economy in man during a hyponitrogenous diet.

Healthy male volunteers (n = 12) were given a normocaloric hyponitrogenous diet for a conditioning period of 7 days. Thereafter they were blindly randomized to receive daily injections of methionyl recombinant human growth hormone (met-hGH) 0.06 IU/kg or saline during a second week of hyponitrogenous nutrition. The met-hGH group showed a lower urinary urea excretion and a lower serum concentration of urea as compared with the control group. In skeletal muscle, the polyribosome concentration, indicative of muscle protein synthesis, as well as the concentrations of glutamine, alanine, aspartate, serine, and threonine, decreased in the control group, whereas no such changes were seen in the met-hGH-treated group. Since provision of met-hGH prevented protein catabolism in muscle and improved whole body nitrogen economy, investigations of the possible beneficial effects of met-hGH to prevent skeletal muscle vast after surgical trauma are advocated.

Transcription of rat liver chromatin by Escherichia coli RNA polymerase: template properties after protein restriction.

Transcription was determined in liver chromatin from rats fed for 6 days, an optimal (20%) or suboptimal (3%) amount of high-quality protein. Transcription by Escherichia coli RNA polymerase (EC 2.7.7.6) was lower after prolonged incubation with chromatin from rats fed 3% as compared with 20% protein. Differences were detected in the transcripts of the two types of chromatin after analysis by sucrose density gradient centrifugation. But no measurable differences were found in the melting profiles at low ionic strength of the two chromatin preparations. Transcription per milligram chromatin DNA was 25-fold higher using E. coli RNA polymerase instead of rat liver RNA polymerase II. The use of UTP as radioactive precursor in the absence of ATP, GTP and CTP resulted in a low labelling of RNA. One [lambda32P]UTP nucleotide was incorporated/8 UMP nucleotides. The product obtained was sensitive to ribonuclease treatment. In the presence of ATP, GTP and CTP [lambda-32P]UTP nucleotide incorporation was reduced and that of UMP nucleotide was increased giving a ratio of 1:188.

The diurnal pattern of protein synthesis in human skeletal muscle.

The diurnal pattern of protein synthesis in skeletal muscle was studied in relation to the serum concentrations of glucose, insulin and cortisol. Twelve healthy volunteers were given an ordinary hospital diet at 4-hourly intervals. The concentration of insulin showed peaks in response to food intake, and that of cortisol decreased over the day. Muscle biopsies were taken 30 min before the first food intake and 1.5 or 3.5 h after a meal. Irrespective of the time intervals after food intake, the total ribosome concentration, percentage amount of polyribosomes and polyribosome concentrations remained unaltered, indicating that there were no alterations in the capacity for protein synthesis accompanying the feeding schedule. The results suggest that a muscle sample taken in the morning after an overnight fast is representative of the protein synthesis during the day as assessed by the ribosome concentration and the size distribution of ribosomes.

RNA metabolism and poly(A) distribution in mouse liver following administration of dimethylnitrosamine.

Dimethylnitrosamine (DMNA) strongly inhibited RNA synthesis in mouse liver under conditions when the nucleotide pattern, rate of nucleotide synthesis and phosphorylation ratio were unaffected. (An unidentified, probably non-nucleotide, component in the acid-soluble liver fraction was selectively reduced.) The inhibition of RNA synthesis was associated with a decrease in the RNA polymerase activity of isolated liver nuclei, well established already 45 min after DMNA administration. The reduced activity included both Mg2+- and Mn2+/(NH4)2SO4-stimulated polymerase functions. The inhibition in vivo involved the whole complement of RNA, including poly (A)-containing RNA and isolated poly(A) sequences. The transfer of labelled RNA from the nucleus to the cytoplasm was not impaired. There was no detachment of poly(A)-containing RNA from the microsomes, and the proportion of tightly membrane-bound microsomal RNA and poly(A) sequences was not reduced as determined by use of a flotation technique. No breakage or shortening of the poly(A) chains was indicated by sedimentation analysis.

O6-methylguanine-DNA methyltransferase and alkylation of liver DNA in mice exposed to dimethylnitrosamine during dietary deficiency of essential amino acids.

Male NMRI mice were fed a diet containing a complete mixture of amino acids or a mixture deficient in methionine-cysteine or lysine (30% of the control level) for a period of 6 days. During the feeding period all mice received dimethylnitrosamine in the drinking water ad libitum. The exposure averaged 1 mg dimethylnitrosamine/kg body weight and day. The concentration of O6-methylguanine-DNA methyltransferase was measured in liver extracts. It decreased significantly in the methionine-cysteine deficient mice. When DNA from the liver was analyzed for alkylated purine bases the mice received a single dose of 14C-labeled dimethylnitrosamine (0.5 or 1 mg/kg body weight) at 120 min before sacrifice. The concentration of O6-methylguanine increased significantly over the control level upon feeding the deficient diets and was restored to the concentration of the controls by refeeding lysine for 2 days following 6 days of lysine deficiency. The increased ratio of O6-methylguanine to N-7-methylguanine indicated that methylation of guanine in the N-7 position was not subject to variation by the intake of dimethylnitrosamine during the dietary deficiencies. The results demonstrate the requirement for a balanced composition of amino acids in the diet to maintain a sufficient concentration of O6-methylguanine-DNA methyltransferase in the cells and thus to permit efficient removal of the methyl group from the O-6 position of guanine in DNA after exposure to dimethylnitrosamine.

Combined effect of dimethylnitrosamine and a lysine-restricted diet on O6-methylguanine-DNA methyltransferase levels in mouse tissues.

O6-Methylguanine is a lesion produced in DNA after exposure of animals to the procarcinogen dimethylnitrosamine. The lesion may lead to mutagenesis or carcinogenesis if not repaired. Repair is accomplished by the protein O6-methylguanine-DNA methyltransferase (MT). The methyl group is transferred to a cysteine residue of the protein, which is not regenerated. In mice, after exposure to alkylating agents, the synthesis of the protein is non-inducible. The inactivation of MT as a result of the transmethylation makes new synthesis of the protein molecules necessary for further dealkylation reactions. Protein synthesis activity correlates well with dietary protein quality. Nutritional conditions of amino acid restriction will limit the number of MT molecules synthesized. Continuous exposure of mice to dimethylnitrosamine will further diminish the pool of the protein. In this study, mice were fed a diet low in lysine and simultaneously given dimethylnitrosamine in the drinking water at concentrations resulting in dosages of zero, 0.4 mg or 1.2 mg/kg body weight/day. After 6 days MT was assayed in liver, kidney and lung. In liver and kidney, lysine restriction provoked a decrease in MT levels per mg of tissue DNA which was intensified by the presence of dimethylnitrosamine in the drinking water. Recovery from lysine restriction with respect to MT levels was achieved within 2 days. In lung, moderate effects on MT levels were observed when dietary lysine restriction was combined with the highest dosage of dimethylnitrosamine used (1.2 mg/kg body weight/day). The data strongly emphasize the importance of an adequate amino acid mixture in the diet, to support protein synthesis and to allow for high MT levels and repair of DNA lesions at the O-6 position of guanine during the exposure of the animals to alkylating agents.

Protein synthesis in skeletal muscle in relation to nitrogen balance after abdominal surgery: the effect of total parenteral nutrition.

Protein synthesis in skeletal muscle was studied in patients after elective abdominal surgery, using the concentration and size distribution of ribosomes. The patients were given either an electrolyte solution or total parenteral nutrition postoperatively. The analyses were performed on muscle biopsy specimens taken prior to surgery and on days 1 and 3 following surgery. The percentage content of polyribosomes, total ribosome concentration and the polyribosome concentrations per milligram of tissue DNA were determined. Elective abdominal surgery caused a significant decrease (p less than 0.05) in the three variables. Total parenteral nutrition did not prevent the decrease. The negative nitrogen balance was significantly improved by total parenteral nutrition, but it did not reach equilibrium. The results show that total parenteral nutrition given postoperatively did not maintain protein synthesis activity in skeletal muscle. The improvement in nitrogen balance suggests that amino acids are utilized by tissues other than skeletal muscle.

Role of glutamine and its analogs in posttraumatic muscle protein and amino acid metabolism.

Skeletal muscle protein catabolism following trauma has until recently not been possible to counteract by intravenous nutritional means. The obligatory loss of nitrogen with concomitant reduction of skeletal muscle protein synthesis is also accompanied by a decrease of muscle free glutamine, the extent of which is proportional to the muscle protein catabolism. Serving as a human model of surgical trauma, patients undergoing elective cholecystectomy were given total parenteral nutrition including additions of either glutamine or its analogs (ornithine-alpha-ketoglutarate, alpha-ketoglutarate, or alanylglutamine) during 3 postoperative days. The polyribosome concentration and the intracellular glutamine concentration in skeletal muscle, as well as nitrogen balance, showed a less pronounced skeletal muscle catabolism in these groups than when conventional total parenteral nutrition was given. It is concluded that a support of either glutamine or its carbon skeleton, alpha-ketoglutarate, counteracts the postoperative fall of muscle free glutamine and of muscle protein synthesis. Furthermore, statistical correlations could be shown between the changes of muscle glutamine and muscle protein synthesis and the postoperative nitrogen losses.