Differential effects of long-term leucine infusion on tissue protein synthesis in neonatal pigs

Leucine is unique among the amino acids in its ability to promote protein synthesis by activating translation initiation via the mammalian target of rapamycin (mTOR) pathway. Previously, we showed that leucine infusion acutely stimulates protein synthesis in fast-twitch glycolytic muscle of neonatal pigs but this response cannot be maintained unless the leucine-induced fall in amino acids is prevented. To determine whether leucine can stimulate protein synthesis in muscles of different fiber types and in visceral tissues of the neonate in the long-term if baseline amino acid concentrations are maintained, overnight fasted neonatal pigs were infused for 24 h with saline, leucine (400 μmol kg⁻¹ h⁻¹), or leucine with replacement amino acids to prevent the leucine-induced hypoaminoacidemia. Changes in the fractional rate of protein synthesis and activation of mTOR, as determined by eukaryotic initiation factor 4E binding protein (4E-BP1) and S6 kinase 1 (S6K1) phosphorylation, in the gastrocnemius and masseter muscles, heart, liver, jejunum, kidney, and pancreas were measured. Leucine increased mTOR activation in the gastrocnemius and masseter muscles, liver, and pancreas, in both the absence and presence of amino acid replacement. However, protein synthesis in these tissues was increased only when amino acids were infused to maintain baseline levels. There were no changes in mTOR signaling or protein synthesis in the other tissues we examined. Thus, long-term infusion of leucine stimulates mTOR signaling in skeletal muscle and some visceral tissues but the leucine-induced stimulation of protein synthesis in these tissues requires sustained amino acid availability.     

Effect of leucine and metabolites of branched chain amino acids on protein turnover in heart.

Leucine, but not isoleucine or valine, inhibited protein degradation and accelerated protein synthesis in hearts perfused with buffer that contained glucose (15 mM) and normal plasma levels of other amino acids, except for the branched chain compounds. Products of leucine, isoleucine, and valine metabolism also inhibited protein degradation and stimulated protein synthesis. These compounds included the transamination and decarboxylation products, as well as acetate, acetoacetate, and propionate. In some, but not all instances, inhibition of degradation and acceleration of synthesis were accompanied by an increase in intracellular leucine. When insulin was added to the perfusate, the rate of degradation was reduced by 40%, but addition of leucine was ineffective in the presence of the hormone. Insulin, leucine (2 mM) and a mixture of branched chain amino acids at normal plasma levels increased latency of cathepsin D in hearts that were perfused with buffer containing glucose. A combination of leucine and insulin increased latency more than either substance alone. These studies indicate that leucine as well as a variety of substrates that are oxidized in the citric acid cycle are involved in regulation of protein turnover in heart muscle.     

Amino acid repletion does not decrease muscle protein catabolism during hemodialysis

Intradialytic protein catabolism is attributed to loss of amino acids in the dialysate. We investigated the effect of amino acid infusion during hemodialysis (HD) on muscle protein turnover and amino acid transport kinetics by using stable isotopes of phenylalanine, leucine, and lysine in eight patients with end-stage renal disease (ESRD). Subjects were studied at baseline (pre-HD), 2 h of HD without amino acid infusion (HD-O), and 2 h of HD with amino acid infusion (HD+AA). Amino acid depletion during HD-O augmented the outward transport of amino acids from muscle into the vein. Increased delivery of amino acids to the leg during HD+AA facilitated the transport of amino acids from the artery into the intracellular compartment. Increase in muscle protein breakdown was more than the increase in synthesis during HD-O (46.7 vs. 22.3%, P < 0.001). Net balance (nmol.min(-1).100 ml (-1)) was more negative during HD-O compared with pre-HD (-33.7 +/- 1.5 vs. -6.0 +/- 2.3, P < 0.001). Despite an abundant supply of amino acids, the net balance (-16.9 +/- 1.8) did not switch from net release to net uptake. HD+AA induced a proportional increase in muscle protein synthesis and catabolism. Branched chain amino acid catabolism increased significantly from baseline during HD-O and did not decrease during HD+AA. Protein synthesis efficiency, the fraction of amino acid in the intracellular pool that is utilized for muscle protein synthesis decreased from 42.1% pre-HD to 33.7 and 32.6% during HD-O and HD+AA, respectively (P < 0.01). Thus amino acid repletion during HD increased muscle protein synthesis but did not decrease muscle protein breakdown.     

Effects of insulin, glucose, and amino acids on protein turnover in rat diaphragm.

A simple method is described for measuring rates of protein synthesis and degradation in isolated rat diaphragm. Muscles incubated in Krebs-Ringer bicarbonate buffer showed a linear rate of synthesis for 3 hours. At the same time, the muscle released tyrosine and ninhydrin-positive material, primarily amino acids, at a linear rate. This release was not a nonspecific leakage of material from the intracellular pools, but reflected net protein degradation. Tyrosine was chosen for studies of protein turnover, since it rapidly equilibrates between intracellular pools and the medium, it can be measured fluorometrically, and it is neither synthesized nor degraded by this tissue. To follow protein degradation independently of synthesis, muscles were incubated in the presence of cycloheximide. Under these conditions, the amount of tyrosine in the intracellular pools was constant, while the muscle released tyrosine at a linear rate. This tyrosine release was used as a measure of degradation. This preparation was used to study the influence of various factors known to be important for muscle growth on protein synthesis and degradation. Similar effects were obtained with diaphragms of normal and fasted rats although the latter showed decreased synthesis and increased protein degradation. Insulin by itself not only stimulated synthesis but also inhibited protein degradation (even in the presence of cycloheximide). These two effects served to reduce the net release of tyrosine from muscle protein to comparable extents. Effects of insulin on synthesis and degradation were greater when glucose was also present in the medium. Glucose by itself inhibited protein degradation but in the absence of insulin glucose had no significant effect on synthesis. Nevertheless, glucose stimulated incorporation of radioactivive tyrosine into protein, but this effect was due to an increased intracellular specific activity. Unlike glucose neither beta-hydroxybutyrate or octanoic acid had any demonstrable effects on protein degradion. The addition of amino acids at plasma concentrations both promoted protein synthesis and inhibited degradation in the diaphragm. Five times normal plasma concentrations of the amino acids had larger effects. The three branched chain amino acids together stimulated synthesis and reduced degradation, while the remaining plasma amino acids did not affect either process significantly. Thus leucine, isoleucine, and valine appear responsible for the effects of plasma amino or isoleucine and valine together, also were able to inhibit protein degradation and promote synthesis.     

Inhibition of hepatocyte proteolysis and lactate gluconeogenesis by chloroquine

Chloroquine (50 μm) is rapidly taken up by isolated hepatocytes in a temperature-dependent manner. It inhibits glucose synthesis from lactate, but not from pyruvate or dihydroxyacetone. The inhibition is reversed by lysine or ammonia but not by oleate or carnitine. Ammonia inhibits chloroquine uptake by the hepatocytes but lysine does not. Chloroquine also inhibits urea synthesis, the release of ninhydrin-reacting substances, the accumulation of amino acids, and the lactate-dependent accumulation of glutamate. Ethanol oxidation in the presence of lactate is also inhibited, and this too is reversed by lysine. Chloroquine increases the redox state of the cytosolic compartment, as evidenced by lactate-to-pyruvate ratios, of hepatocytes prepared from both 48-h fasted and meal-fed rats. The above findings are consistent with chloroquine entering the lysosomes of the hepatocytes and inhibiting proteolysis by raising the lysosomal pH. Isolated hepatocytes are deficient in amino acids and, chloroquine inhibition of proteolysis prevents replenishment of the amino acid pools. Thus, chloroquine prevents reconstitution of the malate-aspartate shuttle required for the movement of reducing equivalents into the mitochondrion during lactate gluconeogenesis, ethanol oxidation, and glycolysis. The metabolic competency of freshly isolated hepatocytes, therefore, depends on the replenishment of amino acid pools by lysosomal breakdown of endogenous protein. Furthermore, chloroquine uptake may be an index of lysosomal function with isolated hepatocytes.     

Effects of amino acid analogues on protein degradation in isolated rat hepatocytes

Analogues and derivatives of six of the amino acids which most effectively inhibit protein degradation in isolated rat hepatocytes (leucine, asparagine, glutamine, histidine, phenylalanine and tryptophan) were investigated to see if they could antagonize or mimic the effect of the parent compound. No antagonists were found. Amino alcohols and amino acid amides tended to inhibit protein degradation strongly, apparently by a direct lysosomotropic effect as indicated by their ability to cause lysosomal vacuolation. Amino acid alkyl esters and dipeptides inhibited degradation to approximately the same extent as did their parent amino acids, possibly by being converted to free amino acids intracellularly. Of several leucine analogues tested, four (L-norleucine, L-norvaline, D-norleucine and L-allo-isoleucine) were found to be as effective as leucine in inhibiting protein degradation. None of the analogues had any effect on protein synthesis. Since leucine appears to play a unique role as a regulator of bulk autophagy in hepatocytes, the availability of active leucine agonists may help tj elucidate the biochemical mechanism for control of this important process.     

The control of protein degradation in monolayer cultures of cat hepatocytes.

1. Isolated cat hepatocytes were established in monolayer culture, cell proteins labelled with tritiated leucine and the effects of amino acids and hormones on the regulation of intracellular protein breakdown were studied. 2. Mixtures of essential and non-essential amino acids inhibited the breakdown of long-lived protein, but when tested individually, amino acids except for tryptophan were ineffective. 3. The rate of breakdown of short-lived protein was not regulated by amino acids or hormones, a finding which was similar to that in rat liver cells. 4. The known stimulatory hormones of proteolysis in rat liver such as glucagon, dexamethasone and corticosteroids failed to enhance protein degradation in cat liver cells. 5. These results support the contention that the control of protein degradation in the cat is different to that in the rat and these differences may reflect the unusual protein metabolism of the cat.     

Reduced amino acid availability inhibits muscle protein synthesis and decreases activity of initiation factor eIF2B

We have examined the effect of a hemodialysis-induced 40% reduction in plasma amino acid concentrations on rates of muscle protein synthesis and breakdown in normal swine. Muscle protein kinetics were measured by tracer methodology using [(2)H(5)]phenylalanine and [1-(13)C]leucine and analysis of femoral arterial and venous samples and tissue biopsies. Net amino acid release by muscle was accelerated during dialysis. Phenylalanine utilization for muscle protein synthesis was reduced from the basal value of 45 +/- 8 to 25 +/- 6 nmol x min(-1) x 100 ml leg(-1) between 30 and 60 min after start of dialysis and was stimulated when amino acids were replaced while dialysis continued. Muscle protein breakdown was unchanged. The signal for changes in synthesis appeared to be changes in plasma amino acid concentrations, as intramuscular concentrations remained constant throughout. The changes in muscle protein synthesis were accompanied by a reduction or stimulation, respectively, in the guanine nucleotide exchange activity of eukaryotic initiation factor (eIF)2B following hypoaminoacidemia vs. amino acid replacement. We conclude that a reduction in plasma amino acid concentrations below the normal basal value signals an inhibition of muscle protein synthesis and that corresponding changes in eIF2B activity suggest a possible role in mediating the response.     

Influence of amino acid availability on protein turnover in perfused skeletal muscle

The effects of amino acids on protein turnover in skeletal muscle were determined in the perfused rat hemicorpus preparation. Perfusion of preparations from fasted young rats (81±2 g) with medium containing either a complete mixture of amino acids at five times (5×) their normal plasma levels, a mixture of leucine, isoleucine, and valine at 5× or 10× levels, or leucine alone (10×) resulted in a 25–50% increase in muscle protein synthesis and a 30% decrease in protein degradation compared to fasted controls perfused in the absence of exogenously added amino acids. When the branched-chain amino acids were omitted from the complete mixture, the remaining amino acids (5×) had no effect on protein turnover. The complete mixture at 1× levels was also ineffective. Comparison of the effects of amino acids with those of glucose and palmitate indicated that amino acids were not acting by providing substrates for energy metabolism. The stimulatory effect of amino acids on protein synthesis was associated with a facilitated rate of peptide-chain initiation as evidenced by a relative decrease in the level of ribosomal subunits. This response was not as great as that produced by insulin, and the amino acids did not augment the effect of insulin. Although protein synthesis in preparations from fed young rats (130±3 g) was stimulated by the addition of a mixture of the branched-chain amino acids (5×) to about the same extent as that observed in the fasted young rats, protein degradation was not affected. Furthermore, neither synthesis nor degradation were affected in preparations from fasted older rats (203±9 g) suggesting that the age and or nitritional state of the animal may influence the response of skeletal muscle to altered amino acid levels.     

Infulence of hormones and medium composition on the degradation of phosphoenolpyruvate carboxykinase (GTP) and total protein in Reuber H35 cells.

Reuber H35 cells were pulse-labeled with radioactive leucine and the influence of hormones, serum, and amino acids on protein degradation was investigated during a subsequent chase period. Radioactive, immunoprecipitable phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.1.32) had a half-life of 5 to 6 hours which was not influenced by either N6, O2-dibutyryl adenosine 3':5'-monophosphate, dexamethasone, or insulin. The rate of phosphoenolpyruvate carboxykinase degradation was the same under steady state conditions as during the approach to a new steady state following hormonal induction or deinduction of the enzyme. Therefore, hormonal regulation of enzyme activity in vivo is the result of changes in the rate of enzyme synthesis. The rate of proteolysis for total cell proteins was increased under nutritional step-down conditions produced by the removal of serum or amino acids, or both, from the medium. This effect was completely prevented by insulin. Cycloheximide and puromycin, but not actinomycin D or cordycepin, inhibited protein degradation under step-down conditions but did not further decrease the basal rate of proteolysis measured in the presence of either insulin or serum plus amino acids. There was a good correlation between changes in proteolysis produced by serum and amino acids and changes in the degradation rate of phosphoenolpyruvate carboxykinase. Also, inhibition of proteolysis with cycloheximide and puromycin was accompanied by a decrease in the degradation rate for enzyme antigen. It is suggested that nutritional step-down leads either to the synthesis or activation of a proteolytic system.     

Regulation of RNA degradation in cultured rat hepatocytes: Effects of specific amino acids and insulin

The regulation of RNA degradation by specific amino acids and insulin was investigated in cultured rat hepatocytes from fed rats previously injected in vivo with [6-¹⁴C]orotic acid. The effects of three groups of amino acids were compared to those of a complete amino acid mixture. The first one consisted of the eight amino acids (leucine, proline, glutamine, histidine, phenylalanine, tyrosine, methionine, tryptophan) previously found to be particularly effective in the control of proteolysis. The two other groups were defined from our study with single additions of amino acids, one consisting of proline, asparagine, glutamine, alanine, phenylalanine, and leucine and the other including the latter group with serine, histidine, and tyrosine. The results showed that these three groups were able to strongly inhibit deprivation-induced RNA breakdown at one and ten times normal plasma concentrations but to a lower extent than the complete amino acid mixture. Six amino acids (proline, asparagine, glutamine, alanine, phenylalanine, leucine) inhibited individually RNA degradation by more than 20%. However, the deletions of proline, asparagine, glutamine, or alanine from the group of these six amino acids were not followed by a loss of inhibitory effect. On the contrary, an important loss of inhibition was observed when leucine and phenylalanine were deleted. Furthermore, only these two amino acids exhibited an additive inhibitory effect. Thus leucine and phenylalanine could be considered as important inhibitors of RNA breakdown in cultured rat hepatocytes. Finally, insulin which had no significant effect on RNA degradation in the absence of amino acids, was able to potentiate the inhibitory effect of different amino acid groups. © 1993 Wiley-Liss, Inc.     

Protein turnover in pulmonary macrophages. Utilization of amino acids derived from protein degradation.

Conditions were defined under which rates of protein synthesis and degradation could be estimated in alveolar macrophages isolated from rabbits by pulmonary lavage and incubated in the presence of plasma concentrations of amino acids and 5.6 mM-glucose. Phenylalanine was validated as suitable precursor for use in these studies: it was not metabolized appreciably, except in the pathways of protein synthesis and degradation; it entered the cells rapidly; it maintained a stable intracellular concentration; and it was incorporated into protein at measurable rates. When extracellular phenylalanine was raised to a concentration sufficient to minimize dilution of the specific radioactivity of the precursor for protein synthesis with amino acid derived from protein degradation, the specific radioactivity of phenylalanyl-tRNA was only 60% of that of the extracellular amino acid. This relationship was unchanged in cells where proteolysis increased 2.5-fold after uptake and degradation of exogenous bovine serum albumin. In contrast, albumin prevented the decrease in phenylalanine incorporation observed in macrophages deprived of an exogenous source of amino acids. These observations suggested that macrophages preferentially re-utilized amino acids derived from the degradation of endogenous, but not from exogenous (albumin), protein. However, when the extracellular supply of amino acids was restricted, substrates derived from albumin catabolism could support the protein-synthetic pathway.     

Acute supplementation of amino acids increases net protein accretion in IUGR fetal sheep

Placental insufficiency decreases fetal amino acid uptake from the placenta, plasma insulin concentrations, and protein accretion, thus compromising normal fetal growth trajectory. We tested whether acute supplementation of amino acids or insulin into the fetus with intrauterine growth restriction (IUGR) would increase net fetal protein accretion rates. Late-gestation IUGR and control (CON) fetal sheep received acute, 3-h infusions of amino acids (with euinsulinemia), insulin (with euglycemia and euaminoacidemia), or saline. Fetal leucine metabolism was measured under steady-state conditions followed by a fetal muscle biopsy to quantify insulin signaling. In CON, increasing amino acid delivery rates to the fetus by 100% increased leucine oxidation rates by 100%. In IUGR, amino acid infusion completely suppressed fetal protein breakdown rates but increased leucine oxidation rate by only 25%, resulting in increased protein accretion rates by 150%. Acute insulin infusion, however, had very little effect on amino acid delivery rates, fetal leucine disposal rates, or fetal protein accretion rates in CON or IUGR fetuses despite robust signaling of the fetal skeletal muscle insulin-signaling cascade. These results indicate that, when amino acids are given directly into the fetal circulation independently of changes in insulin concentrations, IUGR fetal sheep have suppressed protein breakdown rates, thus increasing net fetal protein accretion.     

Mechanisms involved in the coordinate regulation of mTORC1 by insulin and amino acids

In this study, we explored the coordinate regulation of mTORC1 by insulin and amino acids. Rat livers were perfused with medium containing various concentrations of insulin and/or amino acids. At fasting (1×) or 2× (2×AA) concentrations of amino acids, insulin maximally stimulated Akt phosphorylation but had no effect on global rates of protein synthesis. In the absence of insulin, 4×AA produced a moderate stimulation of protein synthesis and activation of mTORC1. The combination of 4×AA and insulin produced a maximal stimulation of protein synthesis and activation of mTORC1. These effects were accompanied by decreases in raptor and PRAS40 and an increase in RagC associated with mTOR (mammalian target of rapamycin). The studies were extended to a cell culture model in which mTORC1 activity was repressed by deprivation of leucine and serum, and resupplementation with the amino acid and insulin acted in an additive manner to restore mTORC1 activation. In deprived cells, mTORC1 was activated by expressing either constitutively active (ca) Rheb or a caRagB·caRagC complex, and coexpression of the constructs had an additive effect. Notably, resupplementation with leucine in cells expressing caRheb or with insulin in cells expressing the caRagB·caRagC complex was as effective as resupplementation with both leucine and insulin in non-transfected cells. Moreover, changes in mTORC1 activity correlated directly with altered association of mTOR with RagB/RagC, Rheb, raptor, and PRAS40. Overall, the results suggest that amino acids signal through the Rag complex and insulin through Rheb to achieve coordinate activation of mTORC1.     

Leucine incorporation and its potential as a measure of protein synthesis by bacteria in natural aquatic systems

Leucine incorporation was examined as a method for estimating rates of protein synthesis by bacterial assemblages in natural aquatic systems. The proportion of the total bacterial population that took up leucine in three marine environments was high (greater than 50%). Most of the leucine (greater than 90%) taken up was incorporated into protein, and little (less than 20%) was degraded to other amino acids, except in two oligotrophic marine environments. In samples from these two environments, ca. 50% of the leucine incorporated had been degraded to other amino acids, which were subsequently incorporated into protein. The degree of leucine degradation appears to depend on the organic carbon supply, as the proportion of 3H-radioactivity incorporated into protein that was recovered as [3H]leucine after acid hydrolysis increased with the addition of pyruvate to oligotrophic water samples. The addition of extracellular leucine inhibited total incorporation of [14C]pyruvate (a precursor for leucine biosynthesis) into protein. Furthermore, the proportion of [14C]pyruvate incorporation into protein that was recovered as [14C]leucine decreased with the addition of extracellular leucine. These results show that the addition of extracellular leucine inhibits leucine biosynthesis by marine bacterial assemblages. The molar fraction of leucine in a wide variety of proteins is constant, indicating that changes in leucine incorporation rates reflect changes in rates of protein synthesis rather than changes in the leucine content of proteins. The results demonstrate that the incorporation rate of [3H]leucine into a hot trichloroacetic acid-insoluble cell fraction can serve as an index of protein synthesis by bacterial assemblages in aquatic systems.     

Whole body protein kinetics in women: effect of pregnancy and IDDM during anabolic stimulation

The effects of pregnancy and type 1 diabetes [insulin-dependent diabetes mellitus (IDDM)] on protein metabolism are still uncertain. Therefore, six normal and five IDDM women were studied during and after pregnancy, using [(13)C]leucine and [(2)H(5)]phenylalanine with a hyperinsulinemic-euglycemic clamp and amino acid infusion. Fasting total plasma amino acids were lower in pregnancy in normal but not IDDM women (2,631 +/- 427 vs. 2,057 +/- 471 and 2,523 +/- 430 vs. 2,500 +/- 440 micromol/l, respectively). Whole body protein breakdown (leucine) increased in pregnancy [change in normal (delta N) and IDDM women (delta D) 0.59 +/- 0.40 and 0.48 +/- 0.26 g. kg(-1). day(-1), both P < 0.001], whereas reductions in protein breakdown due to insulin/amino acids (delta N -0.57 +/- 0.19, delta D -0.58 +/- 0.20 g. kg(-1). day(-1), both P < 0.001) were unaffected by pregnancy. Protein breakdown in IDDM women was not higher than normal, and neither pregnancy nor type 1 diabetes altered the insulin sensitivity of amino acid turnover. Nonoxidized leucine disposal (protein synthesis) increased in pregnancy (delta N 0.67 +/- 0.45, delta D 0.64 +/- 0.34 g. kg(-1). day(-1), both P < 0.001). Pregnancy reduced the response of phenylalanine hydroxylation to insulin/amino acids in both groups (delta N -1.14 +/- 0.74, delta D -1. 12 +/- 0.77 g. kg(-1). day(-1), both P < 0.05). These alterations may enable amino acid conservation for protein synthesis and accretion in late pregnancy. Well-controlled type 1 diabetes caused no abnormalities in the regulation of basal or stimulated protein metabolism.     

Leucine incorporation and its potential as a measure of protein synthesis by bacteria in natural aquatic systems.

Leucine incorporation was examined as a method for estimating rates of protein synthesis by bacterial assemblages in natural aquatic systems. The proportion of the total bacterial population that took up leucine in three marine environments was high (greater than 50%). Most of the leucine (greater than 90%) taken up was incorporated into protein, and little (less than 20%) was degraded to other amino acids, except in two oligotrophic marine environments. In samples from these two environments, ca. 50% of the leucine incorporated had been degraded to other amino acids, which were subsequently incorporated into protein. The degree of leucine degradation appears to depend on the organic carbon supply, as the proportion of 3H-radioactivity incorporated into protein that was recovered as [3H]leucine after acid hydrolysis increased with the addition of pyruvate to oligotrophic water samples. The addition of extracellular leucine inhibited total incorporation of [14C]pyruvate (a precursor for leucine biosynthesis) into protein. Furthermore, the proportion of [14C]pyruvate incorporation into protein that was recovered as [14C]leucine decreased with the addition of extracellular leucine. These results show that the addition of extracellular leucine inhibits leucine biosynthesis by marine bacterial assemblages. The molar fraction of leucine in a wide variety of proteins is constant, indicating that changes in leucine incorporation rates reflect changes in rates of protein synthesis rather than changes in the leucine content of proteins. The results demonstrate that the incorporation rate of [3H]leucine into a hot trichloroacetic acid-insoluble cell fraction can serve as an index of protein synthesis by bacterial assemblages in aquatic systems.     

Stimulation by glucocorticoids of protein degradation in hepatocyte monolayers.

1. Protein degradation in rat hepatocytes in stationary monolayer culture was measured as release of radioactive trichloroacetic acid-soluble material from intracellular proteins labelled with [3H]leucine. 2. Glucocorticoids, but not other steroids, stimulated protein breakdown in the hepatocyte monolayers. The effects observed were greater when the cells were preincubated with the hormones, indicating that the stimulation was not immediate. In addition, the stimulation by glucocorticoids persisted for up to 4 h after hormone removal. 3. Cycloheximide and the lysosomotropic agents leupeptin and ammonia effectively blocked glucocorticoid stimulation of protein degradation. 4. Insulin blocked dexamethasone stimulation when added at the same time as the steroid, but not when added 3 h later. 5. Stimulation of protein breakdown by dexamethasone was additive with that by glucagon or dibutyryl cyclic AMP, suggesting that its mechanism of action is different from that of the latter two agents. 6. Total activities of several lysosomal enzymes were unaffected under conditions where protein breakdown was stimulated by either glucagon or dexamethasone. 7. It is suggested that, whereas glucagon, dibutyryl cyclic AMP and insulin modulate protein breakdown in these cells via changes in autophagocytosis, the stimulation by glucocorticoids is exerted independently, perhaps by stimulating the synthesis of membrane proteins essential to the autophagic process.     

Effects of starvation for potassium and other inorganic ions on protein degradation and ribonucleic acid synthesis in Escherichia coli.

Starvation of Escherichia coli for potassium, phosphate, or magnesium ions leads to a reversible increase in the rate of protein degradation and an inhibition of ribonucleic acid (RNA) synthesis. In cells deprived of potassium, the breakdown of the more stable cell proteins increased two- to threefold, whereas the hydrolysis of short-lived proteins, both normal ones and analog-containing polypeptides, did not change. The mechanisms initiating the enhancement of proteolysis during starvation for these ions were examined. Upon starvation for amino acids or amino acyl-transfer RNA (tRNA), protein breakdown increases in relA+ (but not relA) cells as a result of the rapid synthesis of guanosine-5'-diphosphate-3'-diphosphate (ppGpp). However, a lack of amino acyl-tRNA does not appear to be responsible for the increased protein breakdown in cells starved for inorganic ions, since protein breakdown increased in the absence of these ions in both relA+ and relA cultures, and since a large excess of amino acids did not affect this response. In bacteria in which energy production is restricted, ppGpp levels also rise, and protein breakdown increases. The ion-deprived cultures did show a 40 to 75% reduction in adenosine-5'-triphosphate levels,l similar to that seen upon glucose starvation. However, this decrease in ATP content does not appear to cause the increase in protein breakdown or lead to an accumulation of ppGpp. No consistent change in intracellular ppGpp levels was found in relA+ or relA cells starved for these ions. In addition, in relX mutants, removal of these ions led to accelerated protein degradation even though relX cells are unable to increase ppGpp levels or proteolysis when deprived of a carbon source. In the potassium-, phosphate-, and magnesium-deprived cultures, the addition of choramphenicol or tetracycline caused a reduction in protein breakdown toward basal levels. Such findings, however, do not indicate that protein synthesis is essential for the enhancement of protein degradation, since blockage of protein synthesis by inactivation of a temperature-sensitive valyl-tRNA synthetase did not restore protein catabolism to basal levels. These various results and related studies suggest that the mechanism for increased protein catabolism on starvation for inorganic ions differs from that occurring upon amino acid or arbon deprivation and probably involves an enhanced susceptibility of various cell proteins (especially ribosomal proteins) to proteolysis.