The response of weaned piglets to dietary valine and leucine

Valine (Val) is considered to be the fifth-limiting amino acid in a maize–soyabean meal diet for pigs. Excess leucine (Leu) levels often occur in commercial diets, which may attenuate the effect of Val deficiency because of an increased oxidation of Val. The objective of the present experiment was to determine the effect of increasing concentrations of Leu on the response of young piglets to dietary Val. In all, 75 Large White×Landrace entire male pigs, 44 days of age and with a mean starting weight of 13.5 kg, were used. Three of these were sacrificed at the start to determine their mean initial chemical composition. A summit feed first limiting in Val was serially diluted with a non-protein diluent to produce a series of five digestible Val concentrations of 11.9, 10.1, 8.3, 6.6 and 4.8 g/kg, with a sixth treatment being added to test that the feeds were limiting in Val. Three identical Val series, each with six levels of Val, were supplemented with increasing amounts of Leu (23, 45 and 67 g/kg), thus 18 treatments in total. All pigs were killed at the end of the trial after 18 days for analysis of water, protein, lipid and ash in the carcass. The levels of Val and Leu and their interaction significantly influenced all the measurements taken in the trial. Daily gain in liveweight, water and protein, and feed conversion efficiency all increased with dietary Val content, whereas feed intake decreased as both Val and Leu contents increased. The deleterious effect of increased Leu on feed intake and growth was more marked at lower levels of Val. Supplementing the feed with the lowest Val content with additional Val largely overcame the effect of excess Leu. The efficiency of utilisation of Val for protein growth was unaffected by the level of Leu in the feed, the primary response to excess Leu being a reduction in feed intake. An intake of around 9 g Val/day yielded maximal protein growth during the period from 44 to 62 days of age in pigs of the genotype used in this trial.     

In-vivo control of valine and leucine synthesis in the duckweed Spirodela polyrhiza

Duckweed colonies were grown on 1 l of nutrient solution supplied with 10 M l-[¹⁴C]leucine or with 25 M l-[¹⁴C]valine. Under these conditions the exogenously supplied amino acid did not inhibit growth, but caused in the plants a moderately increased pool of that amino acid, which remained essentially constant during the culture period. The effect of the increased pool of valine or leucine on the biosynthesis of these amino acids was determined from isotope dilution in the protein-bound valine and-or leucine. An increase in the leucine pool from 1.1 to 5.0 nmol mg^–1 dry weight resulted in a 21% reduction of metabolite flow through the common part of the valine-leucine biosynthetic pathway; leucine synthesis was reduced by 35%, but valine synthesis by only 5% and isoleucine synthesis was apparently unaffected. An increase in the valine pool from 3.2 to 6.6 nmol mg^–1 dry weight reduced the metabolite flow through the valine-leucine pathway by 48%, valine synthesis by 70%, and leucine synthesis from pyruvate by 29%, which was compensated by leucine synthesis from exogenous valine, whereas the synthesis of isoleucine was not changed. It is concluded that the biosynthesis of valine and leucine is mainly controlled by feedback inhibition of acetohydroxyacid synthetase. In vivo, the feedback inhibition can be exerted in such a way that synthesis of acetolactate (the precursor of valine and leucine) is appreciably reduced, whereas synthesis of acetohydroxybutyrate (the isoleucine precursor) is not inhibited.     

Transamination and oxidation of leucine and valine in rat adipose tissue

Leucine was oxidized by rat adipose tissue at a rate which was not limited by the activity of branched chain amino acid transaminase since high concentrations (10 mM) of [1-14C]leucine and its transamination product, alpha-keto[1-14C]isocaproate, were oxidized at similar rates. Despite the apparent abundance of transaminase activity, however, [1-14C]valine was oxidized at only 10 to 25% of the rate of its transamination product, alpha-keto[1-14C]isovalerate. The net rate at which [1-14C] valine was transaminated by intact tissues was estimated as the sum of the rates of 14CO2 production and alpha-ketoiso[1-14C]valerate release into the medium. Transamination did not limit the rate of valine oxidation since valine was transaminated 3 times as fast as it was oxidized. The rate of valine transamination increased 18-fold when its concentration was raised 100-fold, but the fraction of [1-14C]valine oxidized to 14CO2 remained constant over the range of incubation conditions studied. The oxidation/transamination ratio for leucine was also constant and exceeded the oxidation/transamination ratio for valine unless valine oxidation was stimulated, either by the addition of glucose or leucine. Stimulation of valine oxidation did not increase its transamination but reduced the rate at which alpha-ketoisovalerate was released from the tissue. The faster oxidation of alpha-ketoisocaproate than of alpha-ketoisovalerate may be due to the activation of branched chain alpha-keto acid dehydrogenase by alpha-ketoisocaproate, but the alpha-keto acid oxidation rates do not fully account for the faster transamination of leucine than of valine.     

The mitochondrial isovaleryl-coenzyme a dehydrogenase of arabidopsis oxidizes intermediates of leucine and valine catabolism

We recently identified a cDNA encoding a putative isovaleryl-coenzyme A (CoA) dehydrogenase in Arabidopsis (AtIVD). In animals, this homotetrameric enzyme is located in mitochondria and catalyzes the conversion of isovaleryl-CoA to 3-methylcrotonyl-CoA as an intermediate step in the leucine (Leu) catabolic pathway. Expression of AtIVD:smGFP4 fusion proteins in tobacco (Nicotiana tabacum) protoplasts and biochemical studies now demonstrate the in vivo import of the plant isovaleryl-CoA dehydrogenase (IVD) into mitochondria and the enzyme in the matrix of these organelles. Two-dimensional separation of mitochondrial proteins by blue native and SDS-PAGE and size determination of the native and overexpressed proteins suggest homodimers to be the dominant form of the plant IVD. Northern-blot hybridization and studies in transgenic Arabidopsis plants expressing Ativd promoter:gus constructs reveal strong expression of this gene in seedlings and young plants grown in the absence of sucrose, whereas promoter activity in almost all tissues is strongly inhibited by exogeneously added sucrose. Substrate specificity tests with AtIVD expressed in Escherichia coli indicate a strong preference toward isovaleryl-CoA but surprisingly also show considerable activity with isobutyryl-CoA. This strongly indicates a commitment of the enzyme in Leu catabolism, but the activity observed with isobutyryl-CoA also suggests a parallel involvement of the enzyme in the dehydrogenation of intermediates of the valine degradation pathway. Such a dual activity has not been observed with the animal IVD and may suggest a novel connection of the Leu and valine catabolism in plants.     

The use of PNAs and their derivatives in mitochondrial gene therapy

Summary Human mitochondria contain their own genome, mtDNA. This small molecule encodes 24 RNA species and 13 polypeptides, which are essential components of the mitochondrial respiratory chain. The mitochondrial genome is present in hundreds or thousands of copies in each cell and is believed to turnover throughout the life of the cell. Defects of the mitochondrial genome (mtDNA) cause a variety of multisystemic disorders routinely affecting the muscle and nervous system. There is currently no effective treatment for patients with defects of the mitochondrial genome. In many patients, defective cells harbour two sub-populations of mtDNA (a situation termed heteroplasmy), one being normal, the other containing the pathogenic mutation. The mutated copy is often recessive, with biochemical and clinical defects only becoming apparent when the levels of mutated mtDNA outweigh the normal copies. It has therefore been postulated that by selectively preventing replication of the mutated mtDNA, the normal copy will propagate, restoring biochemical function. The search has therefore been on during recent years to identify an antigenomic molecule that will fulfil this criterion. Following evidence that peptide nucleic acids could selectively inhibit replication of templates carrying a known pathogenic mtDNA mutation in vitro, we report on the progress of this approach and the various modifications that are now being used to improve the efficacy of PNA-based antigenomic inhibition.     

The use of PNAs and their derivatives in mitochondrial gene therapy

Summary Human mitochondria contain their own genome, mtDNA. This small molecule encodes 24 RNA species and 13 polypeptides, which are essential components of the mitochondrial respiratory chain. The mitochondrial genome is present in hundreds or thousands of copies in each cell and is believed to turnover throughout the life of the cell. Defects of the mitochondrial genome (mtDNA) cause a variety of multisystemic disorders routinely affecting the muscle and nervous system. There is currently no effective treatment for patients with defects of the mitochondrial genome. In many patients, defective cells harbour two sub-populations of mtDNA (a situation termed heteroplasmy), one being normal, the other containing the pathogenic mutation. The mutated copy is often recessive, with biochemical and clinical defects only becoming apparent when the levels of mutated mtDNA outweigh the normal copies. It has therefore been postulated that by selectively preventing replication of the mutated mtDNA, the normal copy will propagate, restoring biochemical function. The search has therefore been on during recent years to identify an antigenomic molecule that will fulfil this criterion. Following evidence that peptide nucleic acids could selectively inhibit replication of templates carrying a known pathogenic mtDNA mutation in vitro, we report on the progress of this approach and the various modifications that are now being used to improve the efficacy of PNA-based antigenomic inhibition.     

The use of PNAs and their derivatives in mitochondrial gene therapy

Human mitochondria contain their own genome, mtDNA. This small molecule encodes 24 RNA species and 13 polypeptides, which are essential components of the mitochondrial respiratory chain. The mitochondrial genome is present in hundreds or thousands of copies in each cell and is believed to turnover throughout the life of the cell. Defects of the mitochondrial genome (mtDNA) cause a variety of multisystemic disorders routinely affecting the muscle and nervous system. There is currently no effective treatment for patients with defects of the mitochondrial genome. In many patients, defective cells harbour two sub-populations of mtDNA (a situation termed heteroplasmy), one being normal, the other containing the pathogenic mutation. The mutated copy is often recessive, with biochemical and clinical defects only becoming apparent when the levels of mutated mtDNA outweigh the normal copies. It has therefore been postulated that by selectively preventing replication of the mutated mtDNA, the normal copy will propagate, restoring biochemical function. The search has therefore been on during recent years to identify an antigenomic molecule that will fulfil this criterion. Following evidence that peptide nucleic acids could selectively inhibit replication of templates carrying a known pathogenic mtDNA mutation in vitro,we report on the progress of this approach and the various modificationsthat are now being used to improve the efficacy of PNA-based antigenomic inhibition.