Leucine Restriction Research Articles

Preservation of Liver Protein Synthesis during Dietary Leucine Deprivation Occurs at the Expense of Skeletal Muscle Mass in Mice Deleted for eIF2 Kinase GCN2

In eukaryotic cells, amino acid depletion reduces translation by a mechanism involving phosphorylation of eukaryotic initiation factor-2 (eIF2). Herein we describe that mice lacking the eIF2 kinase, general control nonderepressible 2 (GCN2) fail to alter the phosphorylation of this initiation factor in liver, and are moribund in response to dietary leucine restriction. Wild-type (GCN2(+/+)) and two strains of GCN2 null (GCN2(-/-)) mice were provided a nutritionally complete diet or a diet devoid of leucine or glycine for 1 h or 6 days. In wild-type mice, dietary leucine restriction resulted in loss of body weight and liver mass, yet mice remained healthy. In contrast, a significant proportion of GCN2(-/-) mice died within 6 days of the leucine-deficient diet. Protein synthesis in wild-type livers was decreased concomitant with increased phosphorylation of eIF2 and decreased phosphorylation of 4E-BP1 and S6K1, translation regulators controlled nutritionally by mammalian target of rapamycin. Whereas translation in the liver was decreased independent of GCN2 activity in mice fed a leucine-free diet for 1 h, protein synthesis in GCN2(-/-) mice at day 6 was enhanced to levels measured in mice fed the complete diet. Interestingly, in addition to a block in eIF2 phosphorylation, phosphorylation of 4E-BP1 and S6K1 was not decreased in GCN2(-/-) mice deprived of leucine for 6 days. This suggests that GCN2 activity can also contribute to nutritional regulation of the mammalian target of rapamycin pathway. As a result of the absence of these translation inhibitory signals, liver weights were preserved and instead, skeletal muscle mass was reduced in GCN2(-/-) mice fed a leucine-free diet. This study indicates that loss of GCN2 eIF2 kinase activity shifts the normal maintenance of protein mass away from skeletal muscle to provide substrate for continued hepatic translation.

Leucine Limitation Induces Autophagy and Activation of Lysosome-dependent Proteolysis in C2C12 Myotubes through a Mammalian Target of Rapamycin-independent Signaling Pathway

Loss of muscle mass usually characterizes different pathologies (sepsis, cancer, trauma) and also occurs during normal aging. One reason for muscle wasting relates to a decrease in food intake. This study addressed the role of leucine as a regulator of protein breakdown in mouse C2C12 myotubes and aimed to determine which cellular responses regulate the process. Determination of the rate of protein breakdown indicated that leucine is one key regulator of this process in myotubes because starvation for this amino acid is responsible for 30-40% of the total increase generated by total amino acid starvation. Leucine restriction rapidly accelerates the rate of protein breakdown (+11 to 15% (p < 0.001) after 1 h of starvation) in a dose-dependent manner. By using various inhibitors, evidence is provided that acceleration of protein catabolism results mainly from an induction of autophagy, activation of lysosome-dependent proteolysis, without modification of mRNA levels encoding the lysosomal cathepsins B, L, or D. Those results suggest that autophagy is an essential cellular response for increasing protein breakdown in muscle following food deprivation. Induction of autophagy precedes a decrease in global protein synthesis (-20% to -30% (p < 0.001)) that occurs after 3 h of leucine starvation. Inhibition of the mammalian target of rapamycin (mTOR) activity does not abolish the effect of leucine starvation and the level of phosphorylated ribosomal S6 protein is not affected by leucine withdrawal. These latter data provide clear evidence that the mTOR signaling pathway is not involved in the mediation of leucine effects on both protein synthesis and degradation in C2C12 myotubes.

Isovaleric acidemia: medical and neurodevelopmental effects of long-term therapy.

Nine patients with isovaleric acidemia were treated with a low-protein diet and supplemental glycine for up to 10 years. Carnitine was added to the therapy in four patients. Overall, the treatment was well tolerated, resulting in no significant side effects other than persistent hyperglycinemia. Normal growth was observed in all patients. Of four patients with the chronic phenotype, three, whose treatment was delayed beyond the first year of life, are mentally retarded. Two of five patients with the acute phenotype are retarded. The outcome in these two was complicated in one by neonatal intraventricular hemorrhage and in the other by therapeutic noncompliance. In our patients, only those who were treated successfully from early infancy and had no complications did not develop mental retardation. After initiation of therapy, there was a significant decrease in ketoacidotic attacks requiring hospitalization. Glycine is indicated for the treatment of acute ketoacidosis in these patients; none of the catastrophically ill newborn who received glycine died. The aim of treatment is to reduce the isovaleric acid burden to a minimum. Therapy consisting of leucine restriction with supplemental glycine and carniline should be started as soon as possible after birth.

3‐Hydroxy‐3‐methylglutaric aciduria: Response to carnitine therapy and fat and leucine restriction

A female infant, born to first cousin parents, lapsed into coma with severe metabolic acidosis on day three of life. The gas chromatographic/mass spectrometric urinary organic acid profile showed marked elevation of the leucine metabolites 3-hydroxy-3-methylglutaric, 3-methylglutaconic, 3-methylglutaric and 3-hydroxy-isovaleric acids. Less than 5% of the normal activity of the enzyme 3-hydroxy-3-methylglutaryl CoA lyase was detected in cultured skin fibroblasts. The patient's total and free carnitine was initially low but rose to normal levels after placing her on DL-carnitine (100 mg kg-1 d-1). On a diet providing 87 mg kg-1 d-1 of leucine and only 25% of total calories as fat and 2 g kg-1 d-1 protein, the concentration of the urinary organic acids fell markedly. She is now 15 months old with normal growth and development. This regimen appears effective in the early treatment of 3-hydroxy-3-methylglutaric aciduria.

Iliofemoral venous thrombosis in an infant:Diagnosis by radionuclide venography

tance. Enzyme determinations in the parents of our patient were not allowed. The extreme hypoglycemia observed in our patient may be the result of several factors. This enzyme defect results in impaired ketone body formation, thereby removing the glucose sparing effect of fatty acid oxidation. Leucine does not accumulate, thus a direct lowering effect of this amino acid on blood glucose by hormonal action (insulin) is not to be expected. However, the exact mechanism of so-called leucine-induced hypoglycemia ~ is not well understood and might represent an effect upon blood glucose, similar to that observed in 3-hydroxy-3-methyiglutaric aciduria. We can imagine that a combination of both effects would lead to the extreme hypoglycemia found in our patient. Restriction of leucine from the diet of these patients will probably prevent clinical deterioration. Early recognition of the underlying enzymatic defect is therefore essential. As the enzyme is detectable in fibroblasts, prenatal diagnosis may be possible.

Effect of isoleucine, valine, or leucine starvation on the potential for formation of the branched-chain amino acid biosynthetic enzymes.

The derepression of the isoleucine and valine biosynthetic enzymes in Escherichia coli and Salmonella typhimurium was examined under conditions of restriction of isoleucine, valine, or leucine (the three amino acids needed for multivalent repression of these enzymes). A procedure was used that allowed the measurement of enzyme-forming potential that accumulated during the starvation period, but could not be expressed unless the missing amino acid was supplied. The threonine deaminase (the product of the ilvA gene)-forming potential that accumulated under such conditions was found to be unstable and decayed with a half-life of about 2.5 min (at 37 C). Evidence was obtained that indicates the threonine deaminase-forming potential that accumulates under conditions of isoleucine starvation is in the form of initiated (rifampin-resistant), but uncompleted (actinomycin D-sensitive), messenger ribonucleic acid chains. Furthermore, it appears that a large portion of the threonine deaminase- and dehydrase (the product of the ilvD gene)-forming potential, under such conditions, is in the form of initiated polypeptide chains. Based on these results and results obtained with SuA(-) strains, a model is presented that explains how the second gene (D) in the ilvADE operon can be partially transcribed and translated under conditions in which there are no completed messenger ribonucleic acids for the gene (A) transcribed before it.

Quantitative effects of nutritional essential amino acid deficiency upon immune responses to tumors in mice.

Mice were fed diets deficient in a single essential amino acid, and the primary immune responses to inoculation of allogenic tumor cells was measured by in vitro assay of cellular immunity. Moderate reduction of the amino acids phenylalanine-tyrosine, valine, threonine, methionine-cystine, isoleucine, and tryptophane in the diet produced profound depression of hemagglutinating and blocking antibody responses, although cytotoxic cell-mediated immunity remained intact. These diets had previously been shown to result in a selective depression of tumor growth in mice. Limitation of the amino acids arginine, histidine, and lysine in the diets gave rise to only slight depression of the immune responses. These diets had previously been shown to produce a proportional decrease in both tumor growth and host body weight. Moderate leucine restriction resulted in a paradoxical depression of cytotoxic cell-mediated immunity with little effect on serum blocking activity. Slight increases had previously been noted in the weight of tumors in mice fed leucine-restricted diets. Deficiency or imbalance of essential amino acids in the diet may produce profound depression of immune responses and apparent, marked changes in the immune resistance of the host animal to tumors.

THE DIETARY MANAGEMENT OF LEUCINE-SENSITIVE HYPOGLYCEMIA, WITH REPORT OF A CASE

A dietary regimen, which has been found useful in the management of leucine-sensitive juvenile hypoglycemia, is described. Hypoglycemia is avoided by a combination of leucine restriction and postprandial carbohydrate feedings. Leucine restriction is achieved primarily through limitation of dietary protein, but sufficient protein is supplied to fulfill the minimum requirement. Through the use of this regimen it was possible to achieve satisfactory symptomatic control without the use of corticosteroids.