Essential Branched-chain Amino Acids Research Articles

Akne und Ernährung

In industrialized countries acne presents as an epidemic disease of civilization affecting sebaceous follicles of adolescents and young adults, associated with increased body mass index and insulin resistance. "Western style" diet, characterized by high glycaemic load and increased consumption of insulinotropic milk proteins, plays an important role in acne pathogenesis. On the cellular level, nutrient-derived metabolic signals are sensed by the metabolic transcription factor FoxO1 and integrated by the regulatory kinase mTORC1. mTORC1, the central hub of protein- and lipid biosynthesis, cell growth and proliferation, is activated by insulin, IGF-1 and branched-chain essential amino acids, especially leucine. The understanding of Western diet-mediated nutrient signalling with over-activated mTORC1 offers a reasonable approach for dietary intervention in acne by lowering glycaemic load and consumption of milk and milk products. A suitable diet attenuating increased mTORC1 activity is a Palaeolithic-like diet with reduced intake of sugar, hyperglycaemic grains, milk and milk products but enriched consumption of vegetables and fish.

Biochemical characterization and evaluation of potent inhibitors of the Pseudomonas aeruginosa PA01 acetohydroxyacid synthase

Microbes and plants synthesize essential branched-chain amino acids (BCAAs) such as valine, leucine, and isoleucine via a common biosynthetic pathway in which the first reaction is catalyzed by acetohydroxyacid synthase (AHAS, EC 4.1.3.18). Recently, AHAS was identified as a potential anti bacterial target. To help find an effective inhibitor that could act as an antibacterial compound, we cloned and characterized the catalytic subunit (CSU) of Pseudomonas aeruginosa AHAS, and found four potent inhibitors through chemical library screening. The ilvI gene of P. aeruginosa encodes a 65-kDa AHAS protein, consistent with the size of the purified enzyme on SDS-PAGE. Enzyme kinetics showed that the enzyme has a Km of 14.2 mM and a specific activity of 0.12 U/mg. Enzyme activity was optimum at a temperature of 37 °C and a pH of 7.5. The Kd for thiamine diphosphate (ThDP) was 89.92 ± 17.9 μM, as determined by fluorescence quenching. The cofactor activation constants (Ks) for ThDP and (Kc) for Mg(2+) were 0.6 ± 0.1 and 560.8 ± 7.4 μM, respectively. Further, we determined that AVS2087, AVS2093, AVS2236, and AVS2387 compounds are potent inhibitors of the catalytic subunit of P. aeruginosa AHAS. These compounds inhibit nearly 100% of AHAS activity, with IC50 values of 1.19 μM, 5.0 nM, 25 nM, and 13 nM, respectively. Compound AVS2093 showed growth inhibition with a minimal inhibitory concentration (MIC) of 742.9 μg/ml against P. aeruginosa strain ATCC 9027. Furthermore, these findings were supported by molecular docking studies with the AVS compounds against P. aeruginosa AHAS in which AVS2093 showed minimum binding energy (-7.8 kJ/mol) by interacting with the receptor through a single hydrogen bond of 2.873 Å. Correlation of AVS2093 activity with P. aeruginosa AHAS cell growth inhibition suggested that AHAS might serve as a target protein for the development of novel antibacterial therapeutics. Results of the current study provide an impetus to further evaluate the potency of these inhibitors against pathogenic P. aeruginosa strains in vivo and to design more potent antibacterial agents based on these AVS inhibitors.

B0AT2 (SLC6A15) Is Localized to Neurons and Astrocytes, and Is Involved in Mediating the Effect of Leucine in the Brain

The B0AT2 protein is a product of the SLC6A15 gene belonging to the SLC6 subfamily and has been shown to be a transporter of essential branched-chain amino acids. We aimed to further characterize the B0AT2 transporter in CNS, and to use Slc6a15 knock out (KO) mice to investigate whether B0AT2 is important for mediating the anorexigenic effect of leucine. We used the Slc6a15 KO mice to investigate the role of B0AT2 in brain in response to leucine and in particular the effect on food intake. Slc6a15 KO mice show lower reduction of food intake as well as lower neuronal activation in the ventromedial hypothalamic nucleus (VMH) in response to leucine injections compared to wild type mice. We also used RT-PCR on rat tissues, in situ hybridization and immunohistochemistry on mouse CNS tissues to document in detail the distribution of SLC6A15 on gene and protein levels. We showed that B0AT2 immunoreactivity is mainly neuronal, including localization in many GABAergic neurons and spinal cord motor neurons. B0AT2 immunoreactivity was also found in astrocytes close to ventricles, and co-localized with cytokeratin and diazepam binding inhibitor (DBI) in epithelial cells of the choroid plexus. The data suggest that B0AT2 play a role in leucine homeostasis in the brain.

L-Leucine Alters Pancreatic β-Cell Differentiation and Function via the mTor Signaling Pathway

Leucine (Leu) is an essential branched-chain amino acid, which activates the mammalian target of rapamycin (mTOR) signaling pathway. The effect of Leu on cell differentiation during embryonic development is unknown. Here, we show that Leu supplementation during pregnancy significantly increased fetal body weight, caused fetal hyperglycemia and hypoinsulinemia, and decreased the relative islet area. We also used rat embryonic pancreatic explant culture for elucidating the mechanism of Leu action on β-cell development. We found that in the presence of Leu, differentiation of pancreatic duodenal homeobox-1–positive progenitor cells into neurogenin3-positive endocrine progenitor cells was inefficient and resulted in decreased β-cell formation. Mechanistically, Leu increases the intracellular levels of hypoxia-inducible factor 1-α, a repressor of endocrine fate in the pancreas, by activating the mTOR complex 1 signaling pathway. Collectively, our findings indicate that Leu supplementation during pregnancy could potentially increase the risk of type 2 diabetes mellitus by inhibiting the differentiation of pancreatic endocrine progenitor cells during a susceptible period of fetal life.

Expression of mitochondrial branched-chain aminotransferase and α-keto-acid dehydrogenase in rat brain: implications for neurotransmitter metabolism

In the brain, metabolism of the essential branched chain amino acids (BCAAs) leucine, isoleucine, and valine, is regulated in part by protein synthesis requirements. Excess BCAAs are catabolized or excreted. The first step in BCAA catabolism is catalyzed by the branched chain aminotransferase (BCAT) isozymes, mitochondrial BCATm and cytosolic BCATc. A product of this reaction, glutamate, is the major excitatory neurotransmitter and precursor of the major inhibitory neurotransmitter γ-aminobutyric acid (GABA). The BCATs are thought to participate in a α-keto-acid nitrogen shuttle that provides nitrogen for synthesis of glutamate from α-ketoglutarate. The branched-chain α-keto acid dehydrogenase enzyme complex (BCKDC) catalyzes the second, irreversible step in BCAA metabolism, which is oxidative decarboxylation of the branched-chain α-keto acid (BCKA) products of the BCAT reaction. Maple Syrup Urine Disease (MSUD) results from genetic defects in BCKDC, which leads to accumulation of toxic levels of BCAAs and BCKAs that result in brain swelling. Immunolocalization of BCATm and BCKDC in rats revealed that BCATm is present in astrocytes in white matter and in neuropil, while BCKDC is expressed only in neurons. BCATm appears uniformly distributed in astrocyte cell bodies throughout the brain. The segregation of BCATm to astrocytes and BCKDC to neurons provides further support for the existence of a BCAA-dependent glial-neuronal nitrogen shuttle since the data show that BCKAs produced by glial BCATm must be exported to neurons. Additionally, the neuronal localization of BCKDC suggests that MSUD is a neuronal defect involving insufficient oxidation of BCKAs, with secondary effects extending beyond the neuron.

Amino Acid Metabolism in the Human Fetus at Term: Leucine, Valine, and Methionine Kinetics

Human fetal metabolism is largely unexplored. Understanding how a healthy fetus achieves its fast growth rates could eventually play a pivotal role in improving future nutritional strategies for premature infants. To quantify specific fetal amino acid kinetics, eight healthy pregnant women received before elective cesarean section at term, continuous stable isotope infusions of the essential amino acids [1-13C,15N]leucine, [U-13C5]valine, and [1-13C]methionine. Umbilical blood was collected after birth and analyzed for enrichments and concentrations using mass spectrometry techniques. Fetuses showed considerable leucine, valine, and methionine uptake and high turnover rates. α-Ketoisocaproate, but not α-ketoisovalerate (the leucine and valine ketoacids, respectively), was transported at net rate from the fetus to the placenta. Especially, leucine and valine data suggested high oxidation rates, up to half of net uptake. This was supported by relatively low α-ketoisocaproate reamination rates to leucine. Our data suggest high protein breakdown and synthesis rates, comparable with, or even slightly higher than in premature infants. The relatively large uptakes of total leucine and valine carbon also suggest high fetal oxidation rates of these essential branched chain amino acids.

Novel thromboxane A2 analog-induced IUGR mouse model.

Rodents, particularly rats, are used in the majority of intrauterine growth restriction (IUGR) research. An important tool that is lacking in this field is the ability to impose IUGR on transgenic mice. We therefore developed a novel mouse model of chronic IUGR using U-46619, a thromboxane A2 (TXA2) analog, infusion. TXA2 overproduction is prevalent in human pregnancies complicated by cigarette smoking, diabetes mellitus and preeclampsia. In this model, U-46619 micro-osmotic pump infusion in the last week of C57BL/6J mouse gestation caused maternal hypertension. IUGR pups weighed 15% less, had lighter brain, lung, liver and kidney weights, but had similar nose-to-anus lengths compared with sham pups at birth. Metabolically, IUGR pups showed increased essential branched-chain amino acids. They were normoglycemic yet hypoinsulinemic. They showed decreased hepatic mRNA levels of total insulin-like growth factor-1 and its variants, but increased level of peroxisome proliferator-activated receptor-gamma coactivator-1 alpha. IUGR offspring were growth restricted from birth (P1) through postnatal day 21 (P21). IUGR males caught up with sham males in weight by P28, whereas IUGR females caught up with sham females by P77. IUGR males surpassed sham males in weight by P238. In summary, we have a non-brain sparing IUGR mouse model that has a relative ease of surgical IUGR induction and exhibits features similar to the chronic IUGR offspring of humans and other animal models. As transgenic technology predominates in mice, this model now permits the imposition of IUGR on transgenic mice to interrogate mechanisms of fetal origins of adult disease.

Treatment of Zebrafish Models of Ribosomopathies (Diamond Blackfan Anemia (DBA) and 5q- Syndrome) with L-Leucine Results In An Improvement of Anemia and Developmental Defects: Evidence for a Common Pathway?

AbstractAbstract 195A growing number of disorders associated with impaired ribosome biogenesis and function have been recently recognized and termed ribosomopathies. The first of these to be identified was Diamond Blackfan Anemia (DBA), a congenital autosomal dominant bone marrow failure syndrome of childhood manifested as profound macrocytic anemia. DBA is characterized by enhanced sensitivity of hematopoietic progenitors to apoptosis with evidence of stress erythropoiesis. In addition to bone marrow defects, DBA patients often have craniofacial, genitourinary, cardiac and limb abnormalities and are at an increased risk of developing hematopoietic malignancies and osteosarcoma. Twenty-five percent of patients with DBA have heterozygous mutations in the ribosomal protein S19 (RPS19) gene, which encodes a component of the 40S ribosomal subunit. Additionally, a growing percentage of DBA patients lacking a mutation in the RPS19 gene have been shown to have mutations in other ribosomal protein genes. A second ribosomopathy, 5q- syndrome is a subtype of myelodysplasia, with an erythroid phenotype strikingly similar to DBA. The erythroid phenotype of 5q- syndrome has been recently demonstrated to be attributable to acquired haploinsufficiency of the ribosomal protein RPS14 gene associated with clonal loss of the long arm of chromosome 5. In both DBA and 5q- syndrome, it is unclear why defects in ribosomal proteins, which would be expected to have widespread consequences, have such a specific effect on erythroid maturation. It has been demonstrated that the efficiency of mRNA translation is significantly depressed in cells derived from DBA patients, consistent with a pathogenic ribosomal defect. L-leucine is an essential branched chain amino acid that is known to modulate protein synthesis by enhancing translation. L-leucine has been used to treat DBA patients, and has been shown to lead to improvement in hemoglobin levels and to transfusion independence in some patients. Based on this observation we hypothesized that a common pathway associated with defective mRNA translation underlies both DBA and 5q- MDS, and that activation of translation by L-Leucine would alleviate the anemia associated with both ribosomopathies. In order to address this hypothesis, we have modeled DBA and 5q- MDS in zebrafish embryos using an antisense morpholino appraoach and have demonstrated that similar to the human diseases, Rps19 and Rps14 deficiency lead to defective erythropoiesis and to developmental abnormalities (specific to DBA). Treatment of Rps19 and Rps14 deficient embryos with L-Leucine resulted in partial reversal of both the anemia and the developmental defects. L-Leucine is thought to regulate protein synthesis by acting as a nutrient signal involving the mTOR (mammalian target of rapamycin) signaling pathway. It has been suggested that L-Leucine enhances translation by activating translation initiation factors and by specific up-regulation of the ribosomal protein S6 kinase. Activation of the mTOR pathway is currently being analyzed in the L-Leucine treated Rps19 and Rps14 deficient zebrafish embryos. Additionally, our observations are also being validated in in vitro human models for DBA and 5q-MDS using human hematopoietic progenitor CD34+ cells and shRNAs specific for Rps19 and Rps14 genes followed by L-Leucine treatment. Our studies should provide evidence for activation of pathways involved in mRNA translation and validate the use of the orally administered amino acid L-Leucine as a therapeutic agent in the treatment of DBA as well as 5q- syndrome without the side-effects associated with current therapies for both diseases. Disclosures:No relevant conflicts of interest to declare.

148 Branched Chain Amino Acid Requirements for Term Neonates

Background: Dietary intake should meet the requirement to obtain an optimal growth and neurodevelopment in the neonate. The essential branched chain amino acids (BCAAs), leucine, isoleucine and valine, are mainly used for incorporation into body protein. Current recommended BCAA requirements for infants 0-6 months (respectively 156, 88 and 87 mg•kg−1•d−1, ratio 1.8:1:1) are based on the amino acid content of human milk. However, human milk fluctuates in composition during lactation and even during a feeding, while milk consumption rate varies widely as well. Questions remain on the validity to use mean amino acid composition of human milk to determine requirements. Objectives: To quantify the requirement of leucine, isoleucine and valine in term neonates using the Indicator Amino Acid Oxidation method. Design: Enterally fed term infants received randomly graded intakes of leucine (15-500 mg•kg−1•d−1), isoleucine (5-216 mg•kg−1•d−1) and valine (5-236 mg•kg−1•d−1). Breath samples containing 13CO2 were collected during L-[1-13C]phenylalanine (indicator amino acid) administration, measured by isotope ratio mass spectrometry and analysed using a biphasic regression crossover analysis. Results: 83 term Asian neonates (birth weight: 3.29 ± 0.4 kg, gestational age: 39.4 ± 1.3 wks, postnatal age: 12.6 ± 5.1 d) were included. The mean requirement (at breakpoint) for leucine, isoleucine and valine was respectively 140, 105 and 110 mg•kg−1•d−1 and the upper 95% confidence interval was 240, 152 and 165 mg•kg−1•d−1. Conclusion: The requirements of the individual BCAAs are almost twice the current recommendations. A Leu:lle:Val ratio of 1.3:1:1 is more appropriate in term formula.

Functional fermented whey-based beverage using lactic acid bacteria

Whey protein concentrate (WPC) is employed as functional food ingredient because of its nutritional value and emulsifying properties. However, the major whey protein β-lactoglobulin (BLG) is the main cause of milk allergy. The aim of this study was to formulate a fermented whey beverage using selected lactic acid bacteria and WPC35 (WPC containing 35% of proteins) to obtain a fermented product with low lactose and BLG contents and high essential amino acid concentration. Cell viability, lactose consumption, lactic acid production, proteolytic activity, amino acid release and BLG degradation by the selected strains Lactobacillus acidophilus CRL 636, Lactobacillus delbrueckii subsp. bulgaricus CRL 656 and Streptococcus thermophilus CRL 804, as single or mixed (SLaB) cultures were evaluated in WPC35 (10%, w/v) incubated at 37 °C for 24 h. Then, the fermented WPC35 was mixed with peach juice and calcium lactate (2%, w/v) and stored at 10 °C for 28 days. During fermentation, single cultures grew 1.7–3.1 log CFU/ml and produced 25.1–95.0 mmol/l of lactic acid as consequence of lactose consumption (14.0–41.8 mmol/l) after 12 h fermentation. L. delbrueckii subsp. bulgaricus CRL 656 was the most proteolytic strain (626 µg/ml Leu) and released the branched-chain essential amino acids Leu (16 μg/ml), Ile (27 μg/ml) and Val (43 μg/ml). All strains were able to degrade BLG in a range of 41–85% after 12 h incubation. The starter culture SLaB grew 3.0 log CFU/ml, showed marked pH reduction, produced 122.0 mmol/l of lactic acid, displayed high proteolytic activity (484 μg/ml Leu) releasing Leu (13 μg/ml), Ile (18 μg/ml) and Val (35 μg/ml), and hydrolyzed 92% of BLG. The addition of calcium lactate to WPC35 maintained the drink pH stable during shelf life; no contamination was detected during this period. After 28 days, a decrease in cell viability of all strains was observed being more pronounced for L. delbrueckii subsp. bulgaricus CRL 656 and L. acidophilus CRL 636 (2.3 and 1.9 log CFU/ml, respectively). The results showed that WPC fermentation by rationally selected lactic acid bacteria might be used for developing functional beverages with improved characteristics such as reduced BLG content and increased branched-chain essential amino acids.

Maternal Low-Protein Diet or Hypercholesterolemia Reduces Circulating Essential Amino Acids and Leads to Intrauterine Growth Restriction

OBJECTIVE—We have examined maternal mechanisms for adult-onset glucose intolerance, increased adiposity, and atherosclerosis using two mouse models for intrauterine growth restriction (IUGR): maternal protein restriction and hypercholesterolemia.RESEARCH DESIGN AND METHODS—For these studies, we measured the amino acid levels in dams from two mouse models for IUGR: 1) feeding C57BL/6J dams a protein-restricted diet and 2) feeding C57BL/6J LDL receptor–null (LDLR−/−) dams a high-fat (Western) diet.RESULTS—Both protein-restricted and hypercholesterolemic dams exhibited significantly decreased concentrations of the essential amino acid phenylalanine and the essential branched chain amino acids leucine, isoleucine, and valine. The protein-restricted diet for pregnant dams resulted in litters with significant IUGR. Protein-restricted male offspring exhibited catch-up growth by 8 weeks of age and developed increased adiposity and glucose intolerance by 32 weeks of age. LDLR−/− pregnant dams on a Western diet also had litters with significant IUGR. Male and female LDLR−/− Western-diet offspring developed significantly larger atherosclerotic lesions by 90 days compared with chow-diet offspring.CONCLUSIONS—In two mouse models of IUGR, we found reduced concentrations of essential amino acids in the experimental dams. This indicated that shared mechanisms may underlie the phenotypic effects of maternal hypercholesterolemia and maternal protein restriction on the offspring.

Amino acid profiles in adults with growth hormone (GH) deficiency before and during GH replacement therapy

Objective GH replacement to growth hormone deficient (GHD) adults improves body composition. In a subset however, lean body mass (LBM) fails to increase despite normalization of IGF-I and amino acid availability could be of importance. We analyzed amino acid (AA) profiles in plasma and erythrocytes (RBC) and associations with LBM, serum IGF-I and IGFBP-1 before and during GH replacement. Design and methods Examinations were performed in 15 GHD patients (six women), aged 34–61 yrs before and after 12 months of GH therapy and in a control group of 20 healthy males aged 31–68 yrs. LBM was measured by dual energy X-ray absorptiometry (DXA), free AAs in plasma and RBC by high performance liquid chromatography and serum IGF-I and IGFBP-1 by in-house RIAs. Setting Tertiary care referral centre. Results At baseline, female GHD patients tended to have lower concentrations of the essential branched – chain AAs isoleucine and leucine, total essential AAs, and of the non-essential AA glutamine than the male patients. Male GHD patients tended to have higher plasma and RBC glutamate than controls. At 12 months, IGF-I had normalized in all but one patient and mean LBM gain was 1.9 ± 0.4 kg. AA levels were unchanged. The change in LBM at 12 months was positively correlated to the ratio between the sum of isoleucine, leucine and valine and baseline LBM kg/m 2 ( r = 0.76, p = 0.001, n = 15). Conclusion Our results suggest that the essential branched-chain amino acids in plasma are important for the LBM response to GH substitution. Our finding has to be confirmed in larger groups of GHD adults before making a proper selection of AAs to be measured in plasma and added as dietary supplement during GH therapy. GH administration did not change AA levels and measurements are not useful for monitoring of GH therapy at the time being.

Protein synthesis is severely diminished following a simulated upper GI bleed in patients with cirrhosis

An upper gastrointestinal (GI) bleed in cirrhotic patients has been hypothesised to induce catabolism due to the absence of the essential branched chain amino acid (BCAA) isoleucine and an abundance of the BCAA leucine in haemoglobin. We tested whether an upper GI bleed produces hypoisoleucinemia via BCAA antagonism and impairs protein synthesis. Isoleucine turnover and oxidation was studied in 5 metabolically stable patients with cirrhosis during a 4-h period of intragastric saline infusion followed by a 4-h period in which an upper GI bleed was simulated by an amino acid solution mimicking haemoglobin. The simulated upper GI bleed induced hypoisoleucinemia (26% of initial values) and an increase in leucine (400%) and valine (350%) concentrations. Isoleucine flux and isoleucine oxidation decreased to a third of initial values following a simulated bleed, but the fraction of isoleucine flux used for oxidation did not change. Consequently, the non-oxidative portion of isoleucine flux, representing protein synthesis, decreased similarly. The present study shows that a simulated upper GI bleed induces hypoisoleucinemia and decreases protein synthesis markedly. The fact that the percentage of isoleucine flux that was oxidized was not influenced by the hypoisoleucinemic state can only be explained by BCAA antagonism.

Is it the low-protein diet or simply the salt restriction?

Dietary factors, such as salt and protein intake, may play an important role in the progression of kidney disease. Consequently, dietary manipulations of these constituents are of interest both in experimental models of kidney disease and in clinical trials with patients with chronic kidney disease to assess whether modification of these exposures will result in a stabilization of disease progression.

Expression of cytosolic branched chain aminotransferase (BCATc) mRNA in the developing mouse brain

Branched-chain aminotransferase (BCAT) catalyzes the transamination of essential branched-chain amino acids (BCAAs: leucine, isoleucine and valine) with α-ketoglutarate. Through this reaction, BCAAs provide nitrogen for the synthesis of glutamate, the predominant excitatory neurotransmitter. Two BCAT isoforms have been identified: one cytosolic (BCATc) and one mitochondrial (BCATm). In adult rodents, BCATc is expressed in a wide variety of structures of the central nervous system (CNS), in neurons. So far, no data were available about the expression of BCATc in the developing CNS. Here, we analyse the expression profile of BCATc mRNA in the mouse brain from embryonic day 12.5 to adult age. BCATc mRNA gradually appears in different brain regions starting from early stages of neural development, and is maintained until adulthood. BCATc mRNA is predominantly present in the cerebral cortex, hippocampus, thalamus, ventral midbrain, raphe, cerebellum and precerebellar system. This study represents the first detailed analysis of BCATc mRNA expression in the developing mouse brain.

Cytosolic branched chain aminotransferase (BCATc) mRNA is up-regulated in restricted brain areas of BDNF transgenic mice

Branched chain aminotransferase (BCAT) catalyzes the transamination of the essential branched chain amino acids (leucine, isoleucine and valine) with α-ketoglutarate. BCAT exists in two isoforms: one cytosolic (BCATc), mainly expressed in the nervous system, and the other mitochondrial (BCATm), present in a greater number of tissues. We previously showed that BCATc mRNA and protein expression in the dorsal lateral geniculate nucleus of the thalamus is up-regulated by exogenous administration of brain-derived neurotrophic factor (BDNF) following lesion of the visual cortex in newborn rats. Here, we analyzed the expression of BCATc mRNA in the brain of transgenic mice overexpressing the rat BDNF cDNA under the control of the α-calcium/calmodulin-dependent kinase II (αCaMKII) promoter. In these animals, BDNF is overexpressed in the telencephalon starting from the second postnatal week. RT-PCR and in situ hybridization experiments showed that BCATc mRNA is overexpressed in restricted regions of the cerebral cortex (parietal area) and hippocampus (hilus and CA3 pyramidal cell layer) of adult BDNF transgenic mice respect to wild-type animals. These differences between wt and BDNF mice were not detected in animals of 1 week of age. These results demonstrate that the expression of the BCATc gene in the brain is specifically regulated by BDNF in a time- and region-dependent fashion.

Proteolytic Mechanisms, Not Malnutrition, Cause Loss of Muscle Mass in Kidney Failure

Hypoalbuminemia and muscle atrophy are frequently found in patients with chronic kidney disease (CKD) and patients being treated by dialysis. These abnormalities are usually attributed to malnutrition, meaning that they are caused by an inadequate diet. However, the evidence indicates that malnutrition is rarely the mechanism causing loss of protein stores. Instead, low values of serum albumin are closely related to the presence of inflammation and loss of muscle mass is attributable to activation of specific proteases. In uremic rodents and patients, the initial step in the loss of muscle protein is an activation of caspase-3. This cleaves the complex structure of muscle, and its action can be detected by the presence of a characteristic 14-kDa actin fragment in the insoluble fraction of muscle. The second step in uremia-induced loss of muscle protein is an activation of the ubiquitin-proteasome system, which rapidly degrades proteins released by caspase-3 cleavage of muscle proteins. Activation of both caspase-3 and the ubiquitin-proteasome system occur when there is suppression of the cellular signaling pathway activated by insulin/insulinlike growth factor 1, the phosphatidylinositol 3-kinase/Akt pathway. A potential therapeutic target for preventing loss of muscle protein is to stimulate activity of this signaling pathway.

Structural Determinants for Branched-chain Aminotransferase Isozyme-specific Inhibition by the Anticonvulsant Drug Gabapentin

This study presents the first three-dimensional structures of human cytosolic branched-chain aminotransferase (hBCATc) isozyme complexed with the neuroactive drug gabapentin, the hBCATc Michaelis complex with the substrate analog, 4-methylvalerate, and the mitochondrial isozyme (hBCATm) complexed with gabapentin. The branched-chain aminotransferases (BCAT) reversibly catalyze transamination of the essential branched-chain amino acids (leucine, isoleucine, valine) to alpha-ketoglutarate to form the respective branched-chain alpha-keto acids and glutamate. The cytosolic isozyme is the predominant BCAT found in the nervous system, and only hBCATc is inhibited by gabapentin. Pre-steady state kinetics show that 1.3 mm gabapentin can completely inhibit the binding of leucine to reduced hBCATc, whereas 65.4 mm gabapentin is required to inhibit leucine binding to hBCATm. Structural analysis shows that the bulky gabapentin is enclosed in the active-site cavity by the shift of a flexible loop that enlarges the active-site cavity. The specificity of gabapentin for the cytosolic isozyme is ascribed at least in part to the location of the interdomain loop and the relative orientation between the small and large domain which is different from these relationships in the mitochondrial isozyme. Both isozymes contain a CXXC center and form a disulfide bond under oxidizing conditions. The structure of reduced hBCATc was obtained by soaking the oxidized hBCATc crystals with dithiothreitol. The close similarity in active-site structures between cytosolic enzyme complexes in the oxidized and reduced states is consistent with the small effect of oxidation on pre-steady state kinetics of the hBCATc first half-reaction. However, these kinetic data do not explain the inactivation of hBCATm by oxidation of the CXXC center. The structural data suggest that there is a larger effect of oxidation on the interdomain loop and residues surrounding the CXXC center in hBCATm than in hBCATc.

Effect of decreased glutamine supply on protein and amino acid metabolism in hepatic tissue. A study using isolated perfused rat liver

Glutamine deficiency, a common finding in severe illness, has a negative influence on immune status, protein metabolism, and disease outcome. In several studies, a close relationship between glutamine, branched-chain amino acid (BCAA), and protein metabolism was demonstrated. The aim of the present study was to investigate the effect of glutamine deficiency on amino acid and protein metabolism in hepatic tissue using a model of isolated perfused rat liver (IPRL). Parameters of protein metabolism and amino acid metabolism were measured using both recirculation and single pass technique with L-[1C]leucine and [1-C]ketoisocaproate (KIC) as a tracer. Glutamine concentration in perfusion solution was 0.5 mmol/L in control and 0 mmol/L in the glutamine-deficient group. The net release of glutamine (about 11 mol/g/h) and higher net uptake of most of the amino acids was observed in the glutamine-deficient group. There was an insignificant effect of lack of glutamine on hepatic protein synthesis, proteolysis, and the release of urea. However, significantly lower release of proteins by the liver perfused with glutamine-deficient solution was observed. The lack of glutamine in perfusion solution caused a significant decrease in leucine oxidation (6.66 1.04 v 13.67 2.38, mol/g dry liver/h, P < .05) and an increase in KIC oxidation (163.7 16.5 v 92.0 12.9 mL/g dry liver/h, P < .05). We conclude that decreased delivery of glutamine to hepatic tissue activates glutamine synthesis, decreases resynthesis of essential BCAA from branched-chain keto acids (BCKA), increases catabolism of BCKA, and has an insignificant effect on protein turnover in hepatic tissue. © 2003 Elsevier Inc. All rights reserved.

Effect of alanyl-glutamine on leucine and protein metabolism in irradiated rats

The mechanism by which glutamine produces a favorable effect in the treatment of sepsis, injury, burns and abdominal irradiation is not completely understood. The main aim of this study was to evaluate the effect of alanyl-glutamine (AlaGln) administration on the metabolism of proteins in irradiated rats. The rats were exposed to whole-body irradiation (8Gy) and then fed intragastrically with a mixture of glucose and amino acids either with AlaGln or without AlaGln. At 48 hours after irradiation, parameters of whole-body protein metabolism and DNA synthesis in intestinal mucosa were investigated using a primed, continuous infusion of [1-14C]leucine and [3H]thymidine. In addition, we evaluated the effect of irradiation and AlaGln on gut morphology, blood count and amino acid concentrations in blood plasma and skeletal muscle. Control rats were not irradiated but were given identical treatment. An increase in whole-body leucine oxidation, and insignificant changes in whole-body proteolysis and in protein synthesis were observed after irradiation. In irradiated rats we observed a decrease in muscle glutamine concentration, a decrease in protein synthesis in jejunum, colon and heart, and an increase in synthesis of proteins of blood plasma and spleen. Morphological examination and measurement of DNA synthesis failed to demonstrate any favorable effect of AlaGln supplementation on irradiated gut. However, administration of AlaGln resulted in a decrease in whole-body proteolysis and leucine oxidation which caused an increase in the fraction of leucine incorporated into the pool of body proteins. We conclude that the data obtained demonstrate that irradiation induces metabolic derangement associated with increased oxidation of essential branched-chain amino acids (valine, leucine and isoleucine) and that these disturbances can be ameliorated by administration of AlaGln.