Leucine Metabolism Research Articles

Costeff optic atrophy syndrome: New clinical case and novel molecular findings

3-Methylglutaconic aciduria (MGA) encompasses a heterogeneous group of disorders, often coinciding with elevated levels of urinary 3-methylglutaric acid. Type I MGA is a disorder of leucine metabolism, while the biological basis for the MGA is unclear for the other types (MGA types II-V). MGA type III (Costeff optic atrophy syndrome, autosomal recessive optic atrophy-3 or optic atrophy plus syndrome, OMIM 258501) is distinguished by early bilateral optic atrophy, later-onset spasticity, extrapyramidal dysfunction, ataxia, and occasional cognitive deficits. It is caused by homozygous mutations in the optic atrophy 3 gene (OPA3). We present a case of a patient with MGA who has infantile-onset optic atrophy, ataxia, extrapyramidal movements and spasticity, but with normal intellect. Sequencing of the patient's DNA revealed a homozygous nonsense mutation c.415C>T (p.Q139X) in exon 2 of transcript 2 of the OPA3 gene, as well as a common silent polymorphism c.231T>C in the same exon. This is the first nonsense mutation found in OPA3. The molecular findings in OPA3 are also reviewed, including mutations in OPA3 that result in autosomal dominant optic atrophy and cataract (ADOAC). The recessive mode of inheritance of MGA type III as a result of the p.Q139X mutation is supported by the carrier status of the unaffected father.

Glial Metabolism of Isoleucine

Isoleucine, together with leucine and valine, constitutes the group of branched-chain amino acids (BCAAs). BCAAs are transported from the blood into the brain parenchyma, where they can serve several distinct functions. Since brain tissue is known to oxidatively metabolize BCAAs to CO(2), they are considered as fuel material in brain energy metabolism. Also, in the case of leucine, cultured astrocytes have been reported to be able to completely oxidize BCAA. While the metabolism of leucine by astroglia-rich primary culture (APC) has already been studied in detail, the metabolic fates of isoleucine and valine in these cells remained to be identified. Therefore, in the present study an NMR analysis was performed of (13)C-labelled metabolites generated in the catabolism of [U-(13)C]Ile by astrocytes and released by them into the incubation medium. APC potently removed isoleucine from the medium and metabolized it. The major isoleucine metabolites released from APC are 2-oxo-3-methylvalerate, 2-methylbutyrate, 3-hydroxy-2-methylbutyrate and propionate. To a lesser extent, APC generate and release also [2,3-(13)C]glutamine, [4,5-(13)C]glutamine and (13)C-labelled isotopomers of lactate and citrate. These results show that APC can release into the extracellular milieu catabolites and several TCA cycle dependent metabolites resulting from the degradation of isoleucine.

Mediobasal hypothalamic leucine metabolism inhibits food intake and leucine activates POMC and LepR neurons

Mediobasal hypothalamic leucine metabolism inhibits food intake and leucine activates POMC and LepR neurons

Glucose and Leucine Metabolism in Lung Tranplanted Patients on Low Dose of Steroids for Immunosuppressive Therapy

Background This study examined the metabolic effects of lung transplantation in patients with end-stage respiratory failure on low dose of steroids for immunosuppressive therapy. Methods We examined 6 patients, including 2 women and 4 men of overall mean age 53 ± 15 years and age at transplantation 34 ± 12 months, receiving cyclosporine 5.73 ± 1.43 mg/kg/d or tacrolimus (FK 506) 4.67 ± 0.58 mg/d, azathioprine 0.47 ± 0.29 mg/kg/d, and prednisone 8.25 mg/d for comparison with 6 healthy subjects, who were selected to be comparable to the recipients in terms of anthropometric features and age. A euglycemic hyperinsulinemic clamp (1 mU/kg/min) associated with infusion of glucose and leucine isotopes was performed with indirect calorimetry. Results Lung transplanted patients showed postabsorptive leucine and free fatty acid metabolism similar to controls. In contrast, there was peripheral insulin resistance with respect to glucose metabolism namely, higher values of glucose and insulin vs controls ( P < .03 and P < .02, respectively). During the clamp the metabolic picture was characterized by a relative insulin resistance with respect to glucose metabolism ( P = .07). Lipid and protein metabolism in the basal and insulin-stimulated conditions were similar to the control group. Conclusions In the basal condition insulin resistance is evident with respect to glucose metabolism. The metabolic picture in lung transplanted patients on low-dose steroid therapy was characterized by normal insulin-stimulated glucose, leucine, and free fatty acid metabolism. The minimal metabolic alterations in these patients were not due to transplantation itself but probably mainly attributable to immunosuppressive therapy.

Studies with leucine, β-hydroxybutyrate and ATP citrate lyase-deficient beta cells support the acetoacetate pathway of insulin secretion

We hypothesized that contrasting leucine with its non-metabolizable analog 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid (BCH) might provide new information about metabolic pathways involved in insulin secretion. Both compounds stimulate insulin secretion by allosterically activating glutamate dehydrogenase, which enhances glutamate metabolism. However, we found that leucine was a stronger secretagogue in rat pancreatic islets and INS-1 cells. This suggested that leucine's metabolism contributed to its insulinotropism. Indeed, we found that leucine increased acetoacetate and was metabolized to CO 2 in pancreatic islets and increased short chain acyl-CoAs (SC-CoAs) in INS-1 cells. We then used the leucine–BCH difference to study the hypothesis that acyl groups derived from secretagogue carbon can be transferred as acetoacetate, in addition to citrate, from mitochondria to the cytosol where they can be converted to SC-CoAs. Since BCH cannot form sufficient acetoacetate from glutamate, transport of any glutamate-derived acyl groups to the cytosol in BCH-stimulated cells must proceed mainly via citrate. In ATP citrate lyase-deficient INS-1 cells, which are unable to convert citrate into cytosolic acetyl-CoA, insulin release by BCH was decreased and adding β-hydroxybutyrate or α-ketoisocaproate, which increases mitochondrial acetoacetate, normalized BCH-induced insulin release. This strengthens the concept that acetoacetate-transferred acyl carbon can be converted to cytosolic SC-CoAs to stimulate insulin secretion.

Impact of Growth Hormone and Dehydroepiandrosterone on Protein Metabolism in Glucocorticoid-Treated Patients

Chronic pharmacological glucocorticoid (GC) use causes substantial morbidity from protein wasting. GH and androgens are anabolic agents that may potentially reverse GC-induced protein loss. Our objective was to assess the effect of GH and dehydroepiandrosterone (DHEA) on protein metabolism in subjects on long-term GC therapy. This was an open, stepwise GH dose-finding study (study 1), followed by a randomized cross-over intervention study (study 2). The studies were performed at a clinical research facility. In study 1, six subjects (age 69+/-4 yr) treated with long-term (>6 months) GCs (prednisone dose 8.3+/-0.8 mg/d) were studied before and after two sequential GH doses (0.8 and 1.6 mg/d) for 2 wk each. In study 2, 10 women (age 71+/-3 yr) treated with long-term GCs (prednisone dose 5.4+/-0.5 mg/d) were studied at baseline and after 2-wk treatment with GH 0.8 mg/d, DHEA 50 mg/d, or GH and DHEA (combination treatment). Changes in whole body protein metabolism were assessed using a 3-h primed constant infusion of 1-[13C]leucine, from which rates of leucine appearance, leucine oxidation, and leucine incorporation into protein were estimated. In study 1, GH 0.8 and 1.6 mg/d significantly reduced leucine oxidation by 19% (P=0.03) and 31% (P=0.02), and increased leucine incorporation into protein by 10% (P=0.13) and 19% (P=0.04), respectively. The lower GH dose did not cause hyperglycemia, whereas GH 1.6 mg/d resulted in fasting hyperglycemia in two of six subjects. In study 2, DHEA did not significantly change leucine metabolism alone or when combined with GH. Blood glucose was not affected by DHEA. GH, at a modest supraphysiological dose of 0.8 mg/d, induces protein anabolism in chronic GC users without causing diabetes. DHEA 50 mg/d does not enhance the effect of GH. GH may safely prevent or reverse protein loss induced by chronic GC therapy.

Expression profiles of the genes associated with metabolism and transport of amino acids and their derivatives in rat liver regeneration

Amino acids (AA) are components of protein and precursors of many important biological molecules. To address effects of the genes associated with metabolism and transport of AA and their derivatives during rat liver regeneration (LR), we firstly obtained the above genes by collecting databases data and retrieving related thesis, and then analyzed their expression profiles during LR using Rat Genome 230 2.0 array. The LR-associated genes were identified by comparing the gene expression difference between partial hepatectomy (PH) and sham-operation (SO) rat livers. It was approved that 134 genes associated with metabolism of AA and their derivatives and 26 genes involved in transport of them were LR-associated. The initially and totally expressing number of these genes occurring in initial phase of LR (0.5-4 h after PH), G0/G1 (4-6 h after PH), cell proliferation (6-66 h after PH), cell differentiation and structure-function reconstruction of liver tissue (72-168 h after PH) were respectively 76, 17, 79, 5 and 162, 89, 564, 195, illustrating that these LR-associated genes were initially expressed mainly in initial stage, and functioned in different phases. Frequencies of up-regulation and down-regulation of them being separately 564 and 357 demonstrated that genes up-regulated outnumbered those down-regulated. Categorization of their expression patterns into 22 types implied the diversity of cell physiological and biochemical activities. According to expression changes and patterns of the above-mentioned genes in LR, it was presumed that histidine biosynthesis in the metaphase and anaphase, valine metabolism in the anaphase, and metabolism of glutamate, glutamine, asparate, asparagine, methionine, alanine, leucine and aromatic amino acid almost were enhanced in the whole LR; as for amino acid derivatives, transport of neutral amino acids, urea, gamma-aminobutyric acid, betaine and taurine, metabolism of dopamine, heme, S-adenosylmethionine, thyroxine, and biosynthesis of hydroxyproline, nitric oxide, orinithine, polyamine, carnitine, selenocysteine were augmented during the entire liver restoration. Above results showed that metabolism and transport of AA and their derivates were necessary in liver regeneration.

Impact of Acute and Chronic Low-Dose Glucocorticoids on Protein Metabolism

High-dose glucocorticoids cause acute protein loss by increasing protein breakdown and oxidation. Whether lower glucocorticoid doses, typical of therapeutic use, induce sustained catabolism has not been studied. Our objective was to assess the effect of acute and chronic therapeutic glucocorticoid doses on protein metabolism. We conducted an open longitudinal and a cross-sectional study at a clinical research facility. Ten healthy subjects were studied before and after a short course of prednisolone (5 and 10 mg/d sequentially for 7 d each). Twelve subjects with inactive polymyalgia rheumatica receiving chronic (>12 months) prednisone (mean = 5.0 +/- 0.8 mg/d) were compared with 12 age- and gender-matched normal subjects. Whole-body protein metabolism was assessed using a 3-h primed constant infusion of 1-[(13)C]leucine, from which rates of leucine appearance (leucine Ra, an index of protein breakdown), leucine oxidation (Lox, index of protein oxidation) and leucine incorporation into protein (LIP, index of protein synthesis) were estimated. Prednisolone induced an acute significant increase in Lox (P = 0.008) and a fall in LIP (P = 0.08) but did not affect leucine Ra. There was no significant difference between the effects of the 5- and 10-mg prednisolone doses on leucine metabolism. In subjects receiving chronic prednisone therapy, leucine Ra, Lox, and LIP were not significantly different from normal subjects. Glucocorticoids stimulate protein oxidation after acute but not chronic administration. This time-related change suggests that glucocorticoid-induced stimulation of protein oxidation does not persist but that a metabolic adaptation occurs to limit protein loss.

Different spectrum of mutations of isovaleryl-CoA dehydrogenase ( IVD) gene in Korean patients with isovaleric acidemia

Isovaleric acidemia (IVA) is an autosomal recessive inborn error of the leucine metabolism that is caused by a deficiency of isovaleryl-CoA dehydrogenase (IVD). Recent application of tandem mass spectrometry to newborn screening has allowed a significant expansion of the recognition of individuals with IVD deficiency. Although many patients have been reported worldwide, there are no genetically confirmed patients in Korea. This study characterizes IVD mutations in seven Korean IVA patients from six unrelated families. Bi-directional sequencing analysis identified two novel variations affecting consensus splice sites (c.144+1G>T in intron 1 and c.457-3_2CA>GG in intron 4) and three novel variations altering coding sequences (c.149G>T; Arg21Leu, c.832A>G; Ser249Gly, and c.1135T>G; Phe350Val). Five patients from four families were found to be compound heterozygotes while two unrelated patients were homozygous for the c.457-3_2CA>GG variation. Reverse-transcription polymerase chain reaction confirmed that both intron variations cause aberrant splicing. Furthermore, analysis of cultured lymphocyte extracts of the seven patients showed no detectable enzyme activity and reduced levels of IVD protein (<10.0% of control) in all samples. These results confirm IVD mutations in Korean patients with IVA and reveal that the mutation spectrum is different from previously reported patients.

Cytosolic and Mitochondrial Malic Enzyme Isoforms Differentially Control Insulin Secretion

In islet beta-cells and INS-1 cells both the high activity of malic enzyme and the correlation of insulin secretion rates with pyruvate carboxylase (PC) flux suggest that a pyruvate-malate cycle is functionally relevant to insulin secretion. Expression of the malic enzyme isoforms in INS-1 cells and rat islets was measured, and small interfering RNA was used to selectively reduce isoform mRNA expression in INS-1 cells to evaluate its impact on insulin secretion. The cytosolic NADP(+)-specific isoform (ME1) was the most abundant, with the mitochondrial isoforms NAD(+)-preferred (ME2) expressed at approximately 50%, and the NADP(+)-specific (ME3) at approximately 10% compared with ME1. Selective reduction (89 +/- 2%) of cytosolic ME1 mRNA expression and enzyme activity significantly reduced glucose (15 mM:41 +/- 6%, p < 0.01) and amino acid (4 mM glutamine +/- 10 mM leucine: 39 +/- 6%, p < 0.01)-stimulated insulin secretion. Selective small interfering RNA reduction (51 +/- 6%) of mitochondrial ME2 mRNA expression did not impact glucose-induced insulin secretion, but decreased amino acid-stimulated insulin secretion by 25 +/- 4% (p < 0.01). Modeling of the metabolism of [U-(13)C]glucose by its isotopic distribution in glutamate indicates a second pool of pyruvate distinct from glycolytically derived pyruvate in INS-1 cells. ME1 knockdown decreased flux of both pools of pyruvate through PC. In contrast, ME2 knockdown affected only PC flux of the pyruvate derived from glutamate metabolism. These results suggest a physiological basis for two metabolically and functionally distinct pyruvate cycles. The cycling of pyruvate by ME1 generates cytosolic NADPH, whereas mitochondrial ME2 responds to elevated amino acids and serves to supply sufficient pyruvate for increased Krebs cycle flux when glucose is limiting.

Branched‐chain amino acid metabolism in higher plants

Valine, leucine and isoleucine contain short branched carbohydrate residues responsible for their classification as branched‐chain amino acids (BCAA). Among the proteinogenic amino acids, BCAA show the highest hydrophobicity and are accordingly the major constituents of transmembrane regions of membrane proteins. BCAA cannot be synthesized by humans and thus belong to the essential amino acids. In contrast, plants are able to synthesize these amino acids de novo and are an important source for these compounds in the human diet. However, BCAA cannot only be synthesized in plants, leucine and probably also valine and isoleucine can also be degraded. Many enzymes operating in turnover are found in mitochondria, while some catabolizing activities are located in peroxisomes. The breakdown of BCAA is physically separated from their biosynthesis in chloroplasts. Additionally, in the order of the Capparales, enzymes of the leucine metabolism seem to be evolutionary related to or may even participate in the methionine chain elongation pathway, the early part of the biosynthesis of aliphatic glucosinolates. In summary, in higher plants a complex network of pathways interferes with the homeostasis of Val, Leu and Ile.

Whole-Body Proteolysis Rate Is Elevated in HIV-Associated Insulin Resistance

Type 2 diabetes is characterized by impaired glucose tolerance (IGT) and insulin resistance with respect to glucose metabolism but not amino acid metabolism. We examined whether whole-body leucine and protein metabolism are dysregulated in HIV-infected individuals with IGT. Glucose and leucine kinetics were measured under fasting insulin conditions and during euglycemic hyperinsulinemia using primed-constant infusions of 2H2-glucose and 13C-leucine in 10 HIV-seronegative control subjects, 16 HIV+ subjects with normal glucose tolerance, and 21 HIV+IGT subjects. Glucose disposal rate during hyperinsulinemia was lower in HIV+IGT than the other two groups. Absolute plasma leucine levels and rate of appearance (whole-body proteolysis) were higher in HIV+IGT at all insulin levels but declined in response to hyperinsulinemia in parallel to those in the other two groups. HIV+IGT had greater visceral adiposity, fasting serum interleukin (IL)-8 and free fatty acid levels, and higher lipid oxidation rates during the clamp than the other two groups. These findings implicate several factors in the insulin signaling pathway, which may be further dysregulated in HIV+IGT, and support the notion that insulin signaling pathways for glucose and leucine metabolism may be disrupted by increased proinflammatory adipocytokines (IL-8) and increased lipid oxidation. Increased proteolysis may provide amino acids for gluconeogenesis, exacerbating hyperglycemia in HIV.

Abstracts for Oral Presentation

Abstracts for Oral Presentation

Renal amino acid, fat and glucose metabolism in type 1 diabetic and non-diabetic humans: effects of acute insulin withdrawal

The aim of this study was to test the hypothesis that type 1 diabetes alters renal amino acid, glucose and fatty acid metabolism. We studied five C-peptide-negative, type 1 diabetic subjects during insulin replacement (glucose 5.6 mmol/l) and insulin deprivation (glucose 15.5 mmol/l) and compared them with six non-diabetic subjects. Leucine, phenylalanine, tyrosine, glucose and palmitate tracers were infused after an overnight fast and samples were obtained from the renal vein, femoral vein and femoral artery. Insulin deprivation significantly increased whole-body fluxes (20-25%) of phenylalanine, tyrosine and leucine, and leucine oxidation (50%). Kidney contributed 5-10% to the whole-body leucine and phenylalanine flux. A net uptake of phenylalanine, conversion of phenylalanine to tyrosine (5 micromol/min) and net release of tyrosine (approximately 5 micromol/min) occurred across the kidney. Whole-body (three-fold) and leg (two-fold) leucine transamination increased but amino acid metabolism in the kidney did not alter with diabetes or insulin deprivation. Insulin deprivation doubled endogenous glucose production, renal glucose production was unaltered by insulin deprivation and diabetes (ranging between 100 and 140 micromol/min). Renal palmitate exchange was unaltered by insulin deprivation. In conclusion, kidney post-absorptively accounts for 5-10% of whole-body protein turnover, 15-20% of leucine transamination and 10-15% of endogenous glucose production, and actively converts phenylalanine to tyrosine. During insulin deprivation, leg becomes a major site for leucine transamination but insulin deprivation does not affect renal phenylalanine, leucine, palmitate or glucose metabolism. Despite its key metabolic role, insulin deprivation in type 1 diabetic patients does not alter many of these metabolic functions.

Isovaleric acidemia: New aspects of genetic and phenotypic heterogeneity

Isovaleric acidemia (IVA) is an autosomal recessive inborn error of leucine metabolism caused by a deficiency of the mitochondrial enzyme isovaleryl-CoA dehydrogenase (IVD) resulting in the accumulation of derivatives of isovaleryl-CoA. It was the first organic acidemia recognized in humans and can cause significant morbidity and mortality. Early diagnosis and treatment with a protein restricted diet and supplementation with carnitine and glycine are effective in promoting normal development in severely affected individuals. Both intra- and interfamilial variability have been recognized. Initially, two phenotypes with either an acute neonatal or a chronic intermittent presentation were described. More recently, a third group of individuals with mild biochemical abnormalities who can be asymptomatic have been identified through newborn screening of blood spots by tandem mass spectrometry. IVD is a flavoenzyme that catalyzes the conversion of isovaleryl-CoA to 3-methylcrotonyl-CoA and transfers electrons to the electron transfer flavoprotein. Human IVD has been purified from tissue and recombinant sources and its biochemical and physical properties have been extensively studied. Molecular analysis of the IVD gene from patients with IVA has allowed characterization of different types of mutations in this gene. One missense mutation, 932C>T (A282V), is particularly common in patients identified through newborn screening with mild metabolite elevations and who have remained asymptomatic to date. This mutation leads to a partially active enzyme with altered catalytic properties; however, its effects on clinical outcome and the necessity of therapy are still unknown. A better understanding of the heterogeneity of this disease and the relevance of genotype/phenotype correlations to clinical management of patients are among the challenges remaining in the study of this disorder in the coming years.

Biochemical characterization of human 3‐methylglutaconyl‐CoA hydratase and its role in leucine metabolism

The metabolic disease 3-methylglutaconic aciduria type I (MGA1) is characterized by an abnormal organic acid profile in which there is excessive urinary excretion of 3-methylglutaconic acid, 3-methylglutaric acid and 3-hydroxyisovaleric acid. Affected individuals display variable clinical manifestations ranging from mildly delayed speech development to severe psychomotor retardation with neurological handicap. MGA1 is caused by reduced or absent 3-methylglutaconyl-coenzyme A (3-MG-CoA) hydratase activity within the leucine degradation pathway. The human AUH gene has been reported to encode for a bifunctional enzyme with both RNA-binding and enoyl-CoA-hydratase activity. In addition, it was shown that mutations in the AUH gene are linked to MGA1. Here we present kinetic data of the purified gene product of AUH using different CoA-substrates. The best substrates were (E)-3-MG-CoA (V(max) = 3.9 U.mg(-1), K(m) = 8.3 microM, k(cat) = 5.1 s(-1)) and (E)-glutaconyl-CoA (V(max) = 1.1 U.mg(-1), K(m) = 2.4 microM, k(cat) = 1.4 s(-1)) giving strong evidence that the AUH gene encodes for the major human 3-MG-CoA hydratase in leucine degradation. Based on these results, a new assay for AUH activity in fibroblast homogenates was developed. The only missense mutation found in MGA1 phenotypes, c.719C>T, leading to the amino acid exchange A240V, produces an enzyme with only 9% of the wild-type 3-MG-CoA hydratase activity.

NMR spectroscopic studies on the late onset form of 3‐methylglutaconic aciduria type I and other defects in leucine metabolism

A diagnosis of 3-methylglutaconic aciduria type I (OMIM: 250950) based on elevated urinary excretion of 3-methylglutaconic acid (3MGA), 3-methylglutaric acid (3MG) and 3-hydroxyisovaleric acid (3HIVA) was made in a 61-year-old female patient presenting with leukoencephalopathy slowly progressing over more than 30 years. The diagnosis was confirmed at the enzymatic and molecular level. In vivo brain MR spectroscopic imaging (MRSI) was performed at 3.0 T, and one-dimensional and two-dimensional in vitro NMR spectroscopy of body fluids of the patient was performed at 11.7 T. Additionally, we measured 1D (1)H-NMR spectra of urine of seven patients with a total of four different inborn errors of leucine metabolism. Increased concentrations of 3HIVA, 3MGA (cis and trans) and 3MG were observed in the NMR spectra of the patient's urine. In the cerebrospinal fluid, the 3HIVA concentration was 10 times higher than in the plasma of the patient and only the cis isomer of 3MGA was observed. In vivo brain MRSI showed an abnormal resonance at 1.28 ppm that may be caused by 3HIVA. Comparison of (1)H-NMR spectra of urine samples from all eight patients studied, representing five different inborn errors of leucine metabolism, showed that each disease has typical NMR characteristics. Our leukoencephalopathy patient suffers from a late-onset form of 3-methylglutaconic aciduria type I. In the literature, only very few adult patients with this conditions have been described, and 3HIVA accumulation in white matter in the brain has not been presented before in these patients. Our data demonstrate that (1)H-NMR spectroscopy of urine can easily discriminate between the known inborn errors of leucine metabolism and provide the correct diagnosis.

A single‐residue mutation, G203E, causes 3‐hydroxy‐3‐methylglutaric aciduria by occluding the substrate channel in the 3D structural model of HMG‐CoA lyase

3-Hydroxy-3-methylglutaric aciduria is a rare autosomal recessive genetic disorder that affects ketogenesis and leucine metabolism. The disease is caused by mutations in the gene coding for 3-hydroxy-3-methylglutaryl-coenzyme A lyase (HL). To date 26 different mutations have been described. A (betaalpha)(8) TIM barrel structure has been proposed for the protein, and almost all missense mutations identified so far localize in the beta sheets that define the inside cavity. We report an Italian patient who bears homozygously a novel HL mutation, c.608G > A (p. G203E) in beta sheet six. A structural model of the mutated protein suggests that glutamic acid 203 impedes catalysis by blocking the entrance to the inner cavity of the enzyme. Loss of functionality has been confirmed in expression studies in E. coli, which demonstrate that the G203E mutation completely abolishes enzyme activity. Beta sheet six and beta sheet two are the two protein regions that accumulate most missense mutations, indicating their importance in enzyme functionality. A model for the mechanism of enzyme function is proposed.

Leucine Regulates Translation Initiation of Protein Synthesis in Skeletal Muscle after Exercise

High-performance physical activity and postexercise recovery lead to significant changes in amino acid and protein metabolism in skeletal muscle. Central to these changes is an increase in the metabolism of the BCAA leucine. During exercise, muscle protein synthesis decreases together with a net increase in protein degradation and stimulation of BCAA oxidation. The decrease in protein synthesis is associated with inhibition of translation initiation factors 4E and 4G and ribosomal protein S6 under regulatory controls of intracellular insulin signaling and leucine concentrations. BCAA oxidation increases through activation of the branched-chain alpha-keto acid dehydrogenase (BCKDH). BCKDH activity increases with exercise, reducing plasma and intracellular leucine concentrations. After exercise, recovery of muscle protein synthesis requires dietary protein or BCAA to increase tissue levels of leucine in order to release the inhibition of the initiation factor 4 complex through activation of the protein kinase mammalian target of rapamycin (mTOR). Leucine's effect on mTOR is synergistic with insulin via the phosphoinositol 3-kinase signaling pathway. Together, insulin and leucine allow skeletal muscle to coordinate protein synthesis with physiological state and dietary intake.

Influence of glucagon on protein and leucine metabolism: A study in fasting man with induced insuiin resistance

The counter-regulatory hormones, including glucagon, may be involved in the generation of postoperative negative nitrogen balance. We examined the influence of glucagon on whole body and forearm muscle protein kinetics, determined by L-[1-13C, 15N]leucine, in two matched groups of healthy fasting subjects. In one study somatostatin alone was infused continuously (0.12 mg h-1) and in another with glucagon (0.04 mg h-1) to generate insulin resistance. Somatostatin infusion increased leucine oxidation (P less than 0.05) and reduced the negative protein balance (P less than 0.01) across the forearm; the 15 per cent decrease in protein breakdown was not significant. Whole body leucine kinetics showed increased flux (P less than 0.05) and synthesis (P less than 0.01) but reduced oxidation (P less than 0.05). Hyperglucagonaemia caused a threefold enhancement of leucine oxidation (P less than 0.02), while the negative protein balance further increased (P less than 0.05) across the forearm. Whole body leucine flux was unchanged; oxidation increased (P less than 0.01) and synthesis decreased (P less than 0.01). These studies confirm that physiological hyperglucagonaemia during insulin resistance is catabolic in the short-term and indicates, for the first time, that glucagon may influence muscle protein metabolism acutely in man. We suggest that therapeutic manoeuvres designed to reduce glucagon levels after surgery may ameliorate protein kinetic abnormalities.