Branched-chain Amino Acids Isoleucine Research Articles

Late-life protein or isoleucine restriction impacts physiological and molecular signatures of aging.

Restricting the intake of protein or the branched-chain amino acid isoleucine promotes healthspan and extends lifespan in young or adult mice. However, their effects when initiated in aged animals are unknown. Here we investigate the consequences of consuming a diet with 67% reduction of all amino acids (low AA) or of isoleucine alone (low Ile), in male and female C57BL/6J.Nia mice starting at 20 months of age. Both dietary regimens effectively promote overall metabolic health without reducing calorie intake. Both low AA and low Ile diets improve aspects of frailty and slow multiple molecular indicators of aging rate; however, the low Ile diet reduces grip strength in both sexes and has mixed, sexually dimorphic effects on the heart. These results demonstrate that low AA and low Ile diets can promote aspects of healthy aging in aged mice and suggest that similar interventions might promote healthy aging in older adults.

Dietary lipid is largely deposited in skin and rapidly affects insulating properties.

Skin has been shown to be a regulatory hub for energy expenditure and metabolism: mutations of skin lipid metabolism enzymes can change the rate of thermogenesis and susceptibility to diet-induced obesity. However, little is known about the physiological basis for this function. Here we show that the thermal properties of skin are highly reactive to diet: within three days, a high fat diet reduces heat transfer through skin. In contrast, a dietary manipulation that prevents obesity accelerates energy loss through skins. We found that skin was the largest target in a mouse body for dietary fat delivery, and that dietary triglyceride was assimilated both by epidermis and by dermal white adipose tissue. Skin from mice calorie-restricted for 3 weeks did not take up circulating lipids and showed a highly depleted stratum corneum. Dietary triglyceride acyl groups persist in skin for weeks after feeding. Using multi-modal lipid profiling, we have implicated both keratinocytes and sebocytes in the altered lipids which correlate with thermal function. In response to high fat feeding, wax diesters and ceramides accumulate, and triglycerides become more saturated. In contrast, in response to the dramatic loss of adipose tissue that accompanies restriction of the branched chain amino acid isoleucine, skin becomes more heat-permeable, resisting changes induced by Western diet feeding, with a signature of depleted signaling lipids. We propose that skin should be routinely included in physiological studies of lipid metabolism, given the size of the skin lipid reservoir and its adaptable functionality.

Dietary isoleucine content defines the metabolic and molecular response to a Western diet.

The amino acid composition of the diet has recently emerged as a critical regulator of metabolic health. Consumption of the branched-chain amino acid isoleucine is positively correlated with body mass index in humans, and reducing dietary levels of isoleucine rapidly improves the metabolic health of diet-induced obese male C57BL/6J mice. However, it is unknown how sex, strain, and dietary isoleucine intake may interact to impact the response to a Western Diet (WD). Here, we find that although the magnitude of the effect varies by sex and strain, reducing dietary levels of isoleucine protects C57BL/6J and DBA/2J mice of both sexes from the deleterious metabolic effects of a WD, while increasing dietary levels of isoleucine impairs aspects of metabolic health. Despite broadly positive responses across all sexes and strains to reduced isoleucine, the molecular response of each sex and strain is highly distinctive. Using a multi-omics approach, we identify a core sex- and strain- independent molecular response to dietary isoleucine, and identify mega-clusters of differentially expressed hepatic genes, metabolites, and lipids associated with each phenotype. Intriguingly, the metabolic effects of reduced isoleucine in mice are not associated with FGF21 - and we find that in humans plasma FGF21 levels are likewise not associated with dietary levels of isoleucine. Finally, we find that foods contain a range of isoleucine levels, and that consumption of dietary isoleucine is lower in humans with healthy eating habits. Our results demonstrate that the dietary level of isoleucine is critical in the metabolic and molecular response to a WD, and suggest that lowering dietary levels of isoleucine may be an innovative and translatable strategy to protect from the negative metabolic consequences of a WD.

In silico screening leads to novel scaffolds with both antifungal and anti-NLRP3 inflammasome activity

Due to structural similarities that exist between established inhibitors of the NLRP3-inflammasome, sulfonylureas Glyburide and MCC-950, and herbicidal-sulfonylureas, that specifically target fungal acetohydroxyacid synthase (AHAS), we sought to determine the potential for compounds to block both inflammation and inhibit fungal growth. In silico screening of ∼250,000 compounds was used to identify a prioritized list of chemical structures capable of inhibiting both targets. Prioritization of the top 1% of scores identified ∼70 compounds with a diverse set of scaffolds for testing in vitro. Selected hits were used to assess anti-inflammatory function in a THP-1 challenge model with LPS+ATP and resulting IC50 values were obtained. MIC and hyphal-growth assays were conducted to determine potential antifungal activity using media depleted of branched chain amino acids isoleucine and valine, to confirm on target AHAS inhibition. Identification of hits that exhibited low micromolar activity for NLRP3 and AHAS inhibition were selected for SAR study. In vitro testing of the analogs along with molecular docking led to increased knowledge for lead optimization of the potential hits. In silico screening has resulted in IC50 (IL-1β release) and MIC50 (fungal growth) values with low μM potency against several Candida species. In vivo validation will further confirm the potential of the scaffolds for further synthetic-modification for the rationale design of novel dual-purpose drugs

Quantitative subcellular acyl-CoA analysis reveals distinct nuclear metabolism and isoleucine-dependent histone propionylation

Quantitative subcellular metabolomic measurements can explain the roles of metabolites in cellular processes but are subject to multiple confounding factors. We developed stable isotope labeling of essential nutrients in cell culture-subcellular fractionation (SILEC-SF), which uses isotope-labeled internal standard controls that are present throughout fractionation and processing to quantify acyl-coenzyme A (acyl-CoA) thioesters in subcellular compartments by liquid chromatography-mass spectrometry. We tested SILEC-SF in a range of sample types and examined the compartmentalized responses to oxygen tension, cellular differentiation, and nutrient availability. Application of SILEC-SF to the challenging analysis of the nuclear compartment revealed a nuclear acyl-CoA profile distinct from that of the cytosol, with notable nuclear enrichment of propionyl-CoA. Using isotope tracing, we identified the branched chain amino acid isoleucine as a major metabolic source of nuclear propionyl-CoA and histone propionylation, thus revealing a new mechanism of crosstalk between metabolism and the epigenome.

1860-P: Alanine Transaminase 2 Is Induced in Diabetic Liver and Contributes to Gluconeogenesis from Amino Acids

Gluconeogenesis from amino acids contributes significantly to hepatic glucose production and hyperglycemia in diabetes. Alanine transaminases (ALT) catalyze the bi-directional interconversion of alanine to pyruvate with concomitant interconversion of α-ketoglutarate to glutamate. ALT1 is cytosolic while ALT2 is localized to the mitochondrial matrix. The mRNA expression and protein abundance of ALT1 and 2 was significantly increased in liver of db/db or diet induced obese mice compared to lean controls. The expression of ALT2 was also increased in obese humans compared to lean controls and was downregulated following bariatric surgery. The increase in hepatic ALT2 expression in diabetic mice was caused by endoplasmic reticulum stress and mediated by activating transcription factor 4 (ATF4). Indeed, inhibition of ATF4 reduced ALT2 expression in liver of db/db mice. Consistent with enhanced use of amino acids in gluconeogenesis, db/db and diet-induced obese mice had elevated blood glucose concentrations following IP administration of L-alanine or glutamine. ALT2 silencing in liver had no effect on glucose concentrations in lean mice, but alleviated alanine-induced hyperglycemia in db/db mice; likely by reducing the incorporation of alanine and/or glutamine into newly synthesized glucose. Plasma alanine tended to rise and glutamate significantly increased in db/db mice with ALT2 silencing whereas concentrations of the branched chain amino acids isoleucine, leucine, and valine decreased or tended to decrease. In summary, our results are consistent with a significant role for ALT2 in hepatic gluconeogenesis from amino acids and in the regulation of blood glucose levels in obesity and diabetes. Disclosure M.R. Martino: None. M. Gutierrez Aguilar: None. K.S. McCommis: None. J. Yoshino: None. B.N. Finck: Advisory Panel; Self; Cirius Therapeutics. Stock/Shareholder; Self; Cirius Therapeutics. Funding National Institutes of Health (R01DK117657)

The Relationship between S. aureus and Branched-Chain Amino Acids Content in Composite Cow Milk.

Simple SummaryStaphylococcus aureus is not only a common cause of bovine mastitis, but also an agent of food poisoning in humans. S. aureus has the ability to produce branched-chain amino acids (BCAAs) under certain nutritional conditions. We show that levels of two BCCA (leucine and isoleucine) are correlated to the load of S. aureus in composite milk samples. The application of the ANOVA and Tukey-Kramer analysis showed statistically significant differences in the content of the BCAAs, isoleucine and leucine, between the two groups based on S. aureus positivity (p < 0.001), while bivariate Pearson correlation analysis showed a strong relationship between S. aureus load and the content of these two BCAAs.The early diagnosis of mastitis is an essential factor for the prompt detection of the animal for further actions. In fact, if not culled, infected cows must be segregated from the milking herd and milked last, or milked with separate milking units. Besides microbiological analysis, the somatic cell count (SCC) commonly used as predictor of intramammary infection, frequently lead to a misclassification of milk samples. To overcome these limitations, more specific biomarkers are continuously evaluated. The total amino acid content increases significantly in mastitic milk compared to normal milk. S. aureus requires branched-chain amino acids (BCAAs—isoleucine, leucine, and valine) for protein synthesis, branched-chain fatty acids synthesis, and environmental adaptation by responding to their availability via transcriptional regulators. The increase of BCAAs in composite milk has been postulated to be linked to mammary infection by S. aureus. The aim of this work is to demonstrate, by a direct ion-pairing reversed-phase method, based on the use of the evaporative light-scattering detector (IP-RP-HPLC-ELSD), applied to 65 composite cow milk samples, a correlation between the concentration of isoleucine and leucine, and S. aureus load. The correlation coefficient, r, was found to be 0.102 for SCC (p = 0.096), 0.622 for isoleucine (p < 0.0001), 0.586 for leucine (p < 0.0001), 0.013 for valine (p = 0.381), and 0.07 for tyrosine (p = 0.034), standing for a positive correlation between S. aureus and isoleucine and leucine concentration. The link between the content of BCAAs, isoleucine and leucine, and udder infection by S. aureus demonstrated with our study has an important clinical value for the rapid diagnosis of S. aureus mastitis in cows.

Evidence for the Accumulation of 3‐Hydroxypropionate and β‐Alanine in Arabidopsis thaliana Seedlings via Isoleucine Degradation

Amino acids serve several cellular processes outside of proteinogenesis that influence the growth and development of the plant, as well as the generation of metabolic energy. Here, we examine the contribution of branched‐chain amino acid degradation to the production of β‐alanine in the model organism, Arabidopsis thaliana. β‐alanine is a key intermediate in the synthesis of the essential molecules vitamin B5 and coenzyme A (CoA). Despite the importance, the current knowledge surrounding β‐alanine lacks strong evidence for the primary biosynthetic pathways in higher plants. Previous studies found that polyamines, uracil, and propionate can all serve as sources for β‐alanine. Given that the catabolism of branched‐chain amino acids (BCAAs) valine and isoleucine result in the production of propionyl‐CoA (a derivative of propionate), we hypothesized that these BCAAs could also serve as a source of β‐alanine. Propionyl‐CoA is typically converted to acetyl‐CoA via β‐oxidation, however, one of the intermediates, malonate semialdehyde, can react to form β‐alanine. To trace the production of β‐alanine from propionyl‐CoA and the BCAA isoleucine, we treated wild‐type and transgenic seedlings with isotopically‐labeled precursors and analyzed extracts using 13C‐NMR and GC‐MS. We observed the production of both β‐alanine and a key intermediate, 3‐hydroxypropionate (3HP) in samples treated with propionate and isoleucine. Our data shows that the fed 13C‐isotopes were efficiently incorporated into and recycled through propionyl‐CoA metabolism, resulting in the production of 13C‐labeled β‐alanine. These findings suggest that while there is evidence for the production of β‐alanine via polyamines, uracil, and propionate in other plants; isoleucine, and possibly valine, can also serve as a precursor to this metabolically important molecule.Support or Funding InformationClare Boothe Luce Undergraduate Scholars program funded by the Henry Luce Foundation (Grant #9601)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Metabolomics approach in the investigation of depression biomarkers in pharmacologically induced immune-related depression.

BackgroundThe aim of this study was to identify previously unrecognised biological pathways and biomarkers that might expand the inflammatory hypothesis of depression.MethodsBroad metabolomics analyses in plasma samples from 31 chronic hepatitis C-infected patients with and without immune-related depression were carried out using the Absolute IDQ p180 kit—a targeted metabolomics approach of combined direct flow injection and liquid chromatography that measures acylcarnitines, amino acids, biogenic amines, glycerophospholipids, sphingolipids, and sugars.ResultsThe measurements showed that the average concentration of the branched-chain amino acid isoleucine was significantly lower in depressive HCV patients in comparison to non-depressive HCV patients [depression group: Median 51.35 (43.4–60.2 μmol/L) vs. Median 62.10 (38.4–81.7 μmol/L); U = -2.958; p = 0.002]. All other amino acids, acylcarnitines, biogenic amines, glycerophospholipids, sphingolipids, sugars, liver enzymes and thyroid levels showed no statistically significant differences.ConclusionsThe results of the present study suggest that the branched-chain amino acid isoleucine might play a role in the pathophysiology of immune-related major depression, which expands existing knowledge about inflammatory hypothesis of depression.

Guanine Limitation Results in CodY-Dependent and -Independent Alteration of Staphylococcus aureus Physiology and Gene Expression.

In Staphylococcus aureus, the global transcriptional regulator CodY modulates the expression of hundreds of genes in response to the availability of GTP and the branched-chain amino acids isoleucine, leucine, and valine (ILV). CodY DNA-binding activity is high when GTP and ILV are abundant. When GTP and ILV are limited, CodY's affinity for DNA drops, altering expression of CodY-regulated targets. In this work, we investigated the impact of guanine nucleotides (GNs) on S. aureus physiology and CodY activity by constructing a guaA null mutant (ΔguaA strain). De novo biosynthesis of guanine monophosphate is abolished due to the guaA mutation; thus, the mutant cells require exogenous guanosine for growth. We also found that CodY activity was reduced when we knocked out guaA, activating the Agr two-component system and increasing secreted protease activity. Notably, in a rich, complex medium, we detected an increase in alternative sigma factor B activity in the ΔguaA mutant, which results in a 5-fold increase in production of the antioxidant pigment staphyloxanthin. Under biologically relevant flow conditions, ΔguaA cells failed to form robust biofilms when limited for guanine or guanosine. Transcriptome sequencing (RNA-Seq) analysis of the S. aureus transcriptome during growth in guanosine-limited chemostats revealed substantial CodY-dependent and -independent alterations of gene expression profiles. Importantly, these changes increase production of proteases and δ-toxin, suggesting that S. aureus exhibits a more invasive lifestyle when limited for guanosine. Further, gene products upregulated under GN limitation, including those necessary for lipoic acid biosynthesis and sugar transport, may prove to be useful drug targets for treating Gram-positive infections.IMPORTANCEStaphylococcus aureus infections impose a serious economic burden on health care facilities and patients because of the emergence of strains resistant to last-line antibiotics. Understanding the physiological processes governing fitness and virulence of S. aureus in response to environmental cues is critical for developing efficient diagnostics and treatments. De novo purine biosynthesis is essential for both fitness and virulence in S. aureus since inhibiting production cripples S. aureus's ability to cause infection. Here, we corroborate these findings and show that blocking guanine nucleotide synthesis severely affects S. aureus fitness by altering metabolic and virulence gene expression. Characterizing pathways and gene products upregulated in response to guanine limitation can aid in the development of novel adjuvant strategies to combat S. aureus infections.

Eliminating Warrantless Assumptions Facilitates Consideration of an Electrostatic Model of Ion-Channel Activation

Models of voltage-sensitive ion channels (VSICs) that don’t specify the way reduction in electric field at threshold activation leads to observed outward S4 motions don’t provide satisfactory explanations of voltage sensing [Bezanilla F (2008) Neuron50:456-468]. Current models depend on unrealistic assumptions: screening of charges by a putative water-filled pore, and simple mechanical devices. A gate, screw, paddle or other everyday device cannot physically explain activation of the highly energetic resting channel, a far-from-equilibrium macromolecular system: The α-helix's hydrogen bonds are too weak to support such rigid structures. The energy required to lengthen the H-bonds is provided when the capacitive energy lost in the voltage reduction to threshold does mechanical work on the channel. Similarities exist between excitable membranes and ferroelectrics [Leuchtag HR (1987) J Theor Biol 127:321-340; 127:341-359; (2008) Voltage-Sensitive Ion Channels, Springer]. The ferroelectricity of excitable membranes must originate in the channels, since VSICs are their active components. In ferroelectrics, the dielectric coefficient ε is a function of the electric field. Electrostatic forces are inversely proportional to ε. Evidence from molecules with the sidechains of the branched-chain amino acids isoleucine, leucine and valine shows that their ε becomes remarkably large in a high electric field [Yoshino K, Sakurai T (1991) in: Goodby JW et al. Ferroelectric Liquid Crystals:317-363], comparable to that in a resting channel. According to the Channel Activation by Electrostatic Repulsion (CAbER) model, ε is high at rest potential and drops on depolarization. The consequently increased electrostatic repulsion between positively charged arginine and lysine residues at activation expands the S4 segments. This outward motion reconfigures the VSIC into an “open” structure with pore-domain sites wide enough to accommodate unhydrated permeant ions that hop stochastically across the channel [Leuchtag HR (2016) Biophys. J. 112(3):544a; 112(3):543a-544a].

Branched-chain amino acids and Alzheimer\u2019s disease: a Mendelian randomization analysis

We conducted a two-sample Mendelian randomization study to test the hypothesis that raised plasma levels of the branched-chain amino acids isoleucine, leucine, and valine are associated with Alzheimer’s disease (AD). From a genome-wide association study of 16,596 individuals of European ancestry, we obtained summary statistics for four independent single nucleotide polymorphisms (SNPs) associated with isoleucine levels and one SNP associated with both leucine and valine levels at genome-wide significance. Summary statistics of the associations of the five SNPs with AD were obtained from the International Genomics of Alzheimer’s Project (17,008 AD cases and 37,154 controls). Based on four SNPs, the odds ratio of AD per genetically predicted one standard deviation higher isoleucine levels was 1.35 (95% CI, 1.08–1.69; p = 0.007). The leucine- and valine-raising allele was not associated with AD (p = 0.46). These data suggest that a genetic predisposition to raised plasma isoleucine levels is positively associated with AD.

What's Wrong with the Hodgkin-Huxley Model? An Exercise in Critical Thinking

Critical thinking is fundamental to science: let us examine an assumption of the Hodgkin-Huxley model of excitable membranes. The circuit elements of the HH model are a capacitance C and parallel ionic conductances g in series with their electromotive forces. HH assumes that C is constant while the gs vary with voltage and time. This assumption, adapted to voltage-sensitive ion channels, places the entire burden of explaining channel function on the gs. While the assumption of constant C—and its dimensionless coefficient, dielectric permittivity ε—is a good approximation for a membrane patch, its application at the macromolecular nanoscale is dubious, considering the ubiquitous presence of branched-chain amino acids—isoleucine, leucine and valine—in the channels. The sidechains of these amino acids (and no others) strongly affect ε in certain molecules with structures resembling channel segments. These ferroelectric liquid crystals, such as 4'-3M2CPOOB, exhibit ε values as high as 3000 in their ferroelectric state. Changes in channel ε affect all electrostatic interactions, including the mutual repulsions between the positively charged residues in S4 segments, key components of voltage sensors. The Channel Activation by Electrostatic Repulsion (CAbER) hypothesis assumes the resting channel, at a high electric field, is in a polar, far-from-equilibrium state with high ε, and that threshold depolarization converts it to a nonpolar state with much lower ε. The consequently increased Coulomb repulsion between arginines and lysines explains the observed outward motion of the S4 segments, which then drives the opening of the pore domain to stochastic ion currents. Does critical thinking support constant-C or CAbER? Problem: Two proton charges on a helix of radius 0.80 nm are 0.45 nm apart axially, with an angular separation of 300°. What is their repulsion energy (kJ/mol) when ε is 500? When it is 4? Further reading: HR Leuchtag, Voltage-Sensitive Ion Channels, Springer, 2008.

Chemical Mechanism of the Branched-Chain Aminotransferase IlvE from Mycobacterium tuberculosis.

The biosynthetic pathway of the branched-chain amino acids is essential for Mycobacterium tuberculosis growth and survival. We report here the kinetic and chemical mechanism of the pyridoxal 5'-phosphate (PLP)-dependent branched-chain aminotransferase, IlvE, from M. tuberculosis (MtIlvE). This enzyme is responsible for the final step of the synthesis of the branched-chain amino acids isoleucine, leucine, and valine. As seen in other aminotransferases, MtIlvE displays a ping-pong kinetic mechanism. pK values were identified from the pH dependence on V as well as V/K, indicating that the phosphate ester of the PLP cofactor, and the α-amino group from l-glutamate and the active site Lys204, play roles in acid-base catalysis and binding, respectively. An intrinsic primary kinetic isotope effect was identified for the α-C-H bond cleavage of l-glutamate. Large solvent kinetic isotope effect values for the ping and pong half-reactions were also identified. The absence of a quininoid intermediate in combination with the Dkobs in our multiple kinetic isotope effects under single-turnover conditions suggests a concerted type of mechanism. The deprotonation of C2 of l-glutamate and the protonation of C4' of the PLP cofactor happen synchronously in the ping half-reaction. A chemical mechanism is proposed on the basis of the results obtained here.

Branched‐chain Amino Acid Catabolism is Required for Muscle Cell Differentiation

The importance of branched‐chain amino acids (BCAAs) in promoting skeletal muscle anabolism has been well studied. BCAAs isoleucine, leucine, and valine have been shown to have a profound effect on activating anabolic signaling pathways in skeletal muscle. This occurs in part by the upregulation in activity of the mammalian target of rapamycin complex‐1 (mTORC1), resulting in increased protein synthesis. However, the regulation of branched‐chain amino acids during the development of muscle remains yet to be elucidated. Here, we studied BCAA metabolism during a 5‐day differentiation of L6 myoblasts. Although no change in intracellular BCAA concentrations was observed during differentiation, L6 cells cultured in the absence of leucine were severely impaired in their ability to differentiate as expression of myosin heavy chain (MHC) was completed abrogated at day 5. Two enzymes which are critical for BCAA metabolism are the branched‐chain amino transferase‐2 enzyme (BCAT2) and the branched chain α‐keto acid dehydrogenase complex (BCKD). BCAT2 catabolizes BCAAs to their corresponding alpha‐keto acids, which are then irreversibly decarboxylated by the BCKD complex. The abundance of BCAT2 did not change during differentiation, whereas levels of the E1α subunit of BCKD was increased 7x on day 5 compared to day 1 (p&lt;0.05). Finally, when either BCAT2 or BCKDE1α was knocked down in the presence of leucine, myoblast differentiation was abrogated (BCKDE1α RNAi: MHC decreased 4.5x from day 3 to day 5 (p&lt;0.05); BCAT2 RNAi: MHC levels completely abolished). Our findings suggest that BCAA catabolism may be a critical process in facilitating muscle differentiation.

The Essentiality of Staphylococcal Gcp Is Independent of Its Repression of Branched-Chain Amino Acids Biosynthesis

Our previous studies revealed that the staphylococcal protein Gcp is essential for bacterial growth; however, the essential function of Gcp remains undefined. In this study, we demonstrated that Gcp plays an important role in the modulation of the branched-chain amino acids biosynthesis pathway. Specifically, we identified that the depletion of Gcp dramatically elevated the production of key enzymes that are encoded in the ilv-leu operon and responsible for the biosynthesis of the branched-chain amino acids isoleucine, leucine, and valine (ILV) using proteomic approaches. Using qPCR and promoter-lux reporter fusions, we established that Gcp negatively modulates the transcription of the ilv-leu operon. Gel-shift assays revealed that Gcp lacks the capacity to bind the promoter region of ilv. Moreover, we found that the depletion of Gcp did not influence the transcription level of CodY, a known repressor of the ilv-leu operon, while induced the transcription of CcpA, a known positive regulator of the ilv-leu operon. In addition, the depletion of Gcp decreased the biosynthesis of N6-threonylcarbamoyladenosine (t6A). To elucidate whether the essentiality of Gcp is attributable to its negative modulation of ILV biosynthesis, we determined the impact of the ilv-leu operon on the requirement of Gcp for growth, and revealed that the deletion of the ilv-leu operon did not affect the essentiality of Gcp. Taken together, our results indicate that the essentiality of Gcp isn’t attributable to its negative regulation of ILV biosynthesis in S. aureus. These findings provide new insights into the biological function of the staphylococcal Gcp.

Metabolic fate of isoleucine in a rat model of hepatic encephalopathy and in cultured neural cells exposed to ammonia

Hepatic encephalopathy is a severe neuropathological condition arising secondary to liver failure. The pathogenesis is not well understood; however, hyperammonemia is considered to be one causative factor. Hyperammonemia has been suggested to inhibit tricarboxylic acid (TCA) cycle activity, thus affecting energy metabolism. Furthermore, it has been suggested that catabolism of the branched-chain amino acid isoleucine may help curb the effect of hyperammonemia by by-passing the TCA cycle block as well as providing the carbon skeleton for glutamate and glutamine synthesis thus fixating ammonia. Here we present novel results describing [U-(13)C]isoleucine metabolism in muscle and brain analyzed by mass spectrometry in bile duct ligated rats, a model of chronic hepatic encephalopathy, and discuss them in relation to previously published results from neural cell cultures. The metabolism of [U-(13)C]isoleucine in muscle tissue was about five times higher than that in the brain which, in turn, was lower than in corresponding cell cultures. However, synthesis of glutamate and glutamine was supported by catabolism of isoleucine. In rat brain, differential labeling patterns in glutamate and glutamine suggest that isoleucine may primarily be metabolized in the astrocytic compartment which is in accordance with previous findings in neural cell cultures. Lastly, in rat brain the labeling patterns of glutamate, aspartate and GABA do not suggest any significant inhibition by ammonia of TCA cycle activity which corresponds well to findings in neural cell cultures. Branched-chain amino acids including isoleucine are used for treating hepatic encephalopathy and the present findings shed light on the possible mechanism involved. The low turn-over of isoleucine in rat brain suggests that this amino acid does not serve the role of providing metabolites pertinent to TCA cycle function and hence energy formation as well as the necessary carbon skeleton for subsequent ammonia fixation in hyperammonemia. The higher metabolism of isoleucine in muscle could, however, contribute to ammonia fixation and thus likely be of value in the treatment of hepatic encephalopathy.

Metabolic effects of amino acid mixtures and whey protein in healthy subjects: studies using glucose-equivalent drinks

Background:Milk protein, in particular the whey fraction, has been shown to display insulinotrophic properties in healthy persons and persons with type 2 diabetes. In parallel to the hyperinsulinemia, a pronounced postprandial rise of certain amino acids and of glucose-dependent insulinotrophic polypeptide (GIP) was observed in plasma.Objective:The objective of the study was to determine to what extent the insulinotrophic properties of whey could be simulated by specific amino acid mixtures.Design:Twelve healthy volunteers were served drinks consisting of pure glucose (reference drink) or glucose supplemented with free amino acids or whey proteins (test drinks).Results:A test drink with the branched-chain amino acids isoleucine, leucine, and valine resulted in significantly higher insulin responses than did the glucose reference. A drink containing glucose and leucine, isoleucine, valine, lysine, and threonine mimicked the glycemic and insulinemic responses seen after whey ingestion. With consumption of this drink, the glucose area under the curve (AUC) was 44% smaller (P< 0.05) and the insulin AUC was 31% larger (NS) than with consumption of the reference drink. With consumption of the whey drink, the AUCs were 56% smaller (glucose; P< 0.05) and 60% larger (insulin; P< 0.05), respectively, than with the reference drink. The whey drink was accompanied by an 80% greater GIP response (P< 0.05), whereas the drinks containing free amino acids did not significantly affect GIP secretion.Conclusion:A mixture of leucine, isoleucine, valine, lysine, and threonine resulted in glycemic and insulinemic responses closely mimicking those seen after whey ingestion in the absence of an additional effect of GIP and glucagon-like peptide 1.

The metabolic role of isoleucine in detoxification of ammonia in cultured mouse neurons and astrocytes

Cerebral hyperammonemia is a hallmark of hepatic encephalopathy, a debilitating condition arising secondary to liver disease. Pyruvate oxidation including tricarboxylic acid (TCA) cycle metabolism has been suggested to be inhibited by hyperammonemia at the pyruvate and α-ketoglutarate dehydrogenase steps. Catabolism of the branched-chain amino acid isoleucine provides both acetyl-CoA and succinyl-CoA, thus by-passing both the pyruvate dehydrogenase and the α-ketoglutarate dehydrogenase steps. Potentially, this will enable the TCA cycle to work in the face of ammonium-induced inhibition. In addition, this will provide the α-ketoglutarate carbon skeleton for glutamate and glutamine synthesis by glutamate dehydrogenase and glutamine synthetase (astrocytes only), respectively, both reactions fixing ammonium. Cultured cerebellar neurons (primarily glutamatergic) or astrocytes were incubated in the presence of either [U- 13C]glucose (2.5 mM) and isoleucine (1 mM) or [U- 13C]isoleucine and glucose. Cell cultures were treated with an acute ammonium chloride load of 2 (astrocytes) or 5 mM (neurons and astrocytes) and incorporation of 13C-label into glutamate, aspartate, glutamine and alanine was determined employing mass spectrometry. Labeling from [U- 13C]glucose in glutamate and aspartate increased as a result of ammonium-treatment in both neurons and astrocytes, suggesting that the TCA cycle was not inhibited. Labeling in alanine increased in neurons but not in astrocytes, indicating elevated glycolysis in neurons. For both neurons and astrocytes, labeling from [U- 13C]isoleucine entered glutamate and aspartate albeit to a lower extent than from [U- 13C]glucose. Labeling in glutamate and aspartate from [U- 13C]isoleucine was decreased by ammonium treatment in neurons but not in astrocytes, the former probably reflecting increased metabolism of unlabeled glucose. In astrocytes, ammonia treatment resulted in glutamine production and release to the medium, partially supported by catabolism of [U- 13C]isoleucine. In conclusion, i) neuronal and astrocytic TCA cycle metabolism was not inhibited by ammonium and ii) isoleucine may provide the carbon skeleton for synthesis of glutamate/glutamine in the detoxification of ammonium.

Intermediate metabolism in association with the amino acid profile during the third trimester of normal pregnancy and diet-controlled gestational diabetes

The aim of this study was to investigate the levels of fasting maternal plasma amino acids in normal pregnant women and compare them with those in gestational diabetes controlled by diet only. We also wished to delineate the alterations occurring in intermediate metabolic pathways in gestational diabetes. Forty-six (64.7%) pregnant women with uncomplicated pregnancy (NP group) and 25 (35.2%) women with gestational diabetes mellitus (GDM group) at 30-33 weeks of gestation participated in the study. Fasting maternal plasma carnitine (total, free, and acyl-carnitine), beta-hydroxybutyrate, free fatty acids, glycosylated hemoglobin, and 21 amino acids were assayed. Fasting carnitine esters exhibited lower levels in the GDM group (P = .03). Higher levels of fasting beta-hydroxybutyrate and free fatty acids (P < .001 and .02, respectively) were observed in the GDM group. Of the 21 amino acids, only methionine, glycine, alanine, citrulline, and ornithine levels were found at significantly higher levels in the NP group (P = .03, P < .001, P = .01, P < .001, and P < .001, respectively). In gestational diabetes, ketogenic amino acids and the branched-chain amino acid isoleucine are released at low rates from skeletal muscle and are mostly catabolized in the liver rather than in peripheral tissues. In contrast, in normal pregnancy proteinolysis is enhanced, and the ketogenic amino acids along with branched-chain amino acids are catabolized in both the liver and peripheral tissues. As a result, ketogenic amino acids are fully oxidized and gluconeogenesis becomes more efficient, whereas urea cycle operates at a higher rate.