Blood Branched-chain Amino Acids Research Articles

Liver regeneration after hepatectomy is significantly suppressed in a muscular atrophy mouse model.

Sarcopenia is a syndrome characterized by progressive and generalized loss of skeletal muscle mass and strength. As reported in previous studies, the loss of skeletal muscle mass is associated with poor liver regeneration after hepatectomy. It is considered important to clarify the effect of sarcopenia on liver regeneration; however, there are no reports about model animals for sarcopenia. We focused on the peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PGC-1α) transgenic mice that overexpressed PGC-1α, specifically for skeletal muscle, and showed significant atrophy of type 2B fiber-rich muscles like sarcopenia. We performed 70% hepatectomy using PGC-1α transgenic mice and examined the liver regeneration rate and the effects of branched-chain amino acids (BCAA) after hepatectomy. Liver regeneration after 70% hepatectomy was significantly suppressed in the PGC-1α transgenic mice. In addition, a decrease in the blood BCAA concentration and a decrease in the liver glycogen content after 70% hepatectomy were observed in the PGC-1α transgenic mice. By administering BCAA before and after surgery, it was clarified that a significant increase in the blood BCAA concentration was observed and the liver regeneration rate was improved in the PGC-1α transgenic mice. BCAA administration may improve the suppression of liver regeneration in patients with sarcopenia.

Mechanism of skeletal muscle atrophy by muscle fiber types in male rats under long-term fasting stress

Fasting induces metabolic changes in muscles, which are differentiated by muscle fiber type. In this study, the mechanism of fasting-induced muscle atrophy in rats was examined to determine the differences between muscle fiber types in energy production. Fasting for 96 h did not alter the weight of the soleus (SOL), a fiber type I muscle, but did significantly reduce the weight of gastrocnemius (GM), a fiber type II muscle. GM, SOL and blood pregnenolone and testosterone levels decreased under fasting, which induced energy deprivation, whereas corticosterone (CORT) levels significantly increased. However, the expression of 3β-HSD and P45011β in GM was unaffected by fasting. The decrease in GM weight may be due to decreased levels of testosterone and reduced synthesis of mammalian target of rapamycin (mTOR). Significant increases in CORT both GM and SOL were associated with increases in the amount of branched-chain amino acids available for energy production. However, decreased levels of mTOR and IGF1 and increased levels of CORT and IL-6 in SOL suggest that GM proteolysis was followed by SOL proteolysis for additional energy production. In conclusion, IGF1 levels decreased significantly in SOL, whereas those of IL-6 significantly increased in SOL and blood but decreased in GM. Blood branched-chain amino acids (BCAA) levels were unaffected due to fasting, whereas an increase was noted in the levels of BCAA in GM and SOL. These results show that fasting for 96 h restricts energy supply, producing fast-twitch muscle atrophy followed by slow-twitch muscle atrophy.

Correlation of Salivary Occult Blood with the Plasma Concentration of Branched-Chain Amino Acids: A Cross-Sectional Study.

Background: Plasma branched-chain amino acids (BCAA) levels are predictors of glycometabolic disorders, leading to diabetes. Microbes, including periodontal pathogens, are thought to be associated with elevated plasma BCAA levels. This study aimed to evaluate the relationship between salivary occult blood (SOB) and plasma BCAA levels in middle-aged Japanese individuals. Methods: Sixty-four Japanese individuals aged ≥ 40 years were recruited for this study, which was conducted in Fukuoka Prefecture, Japan, from August to December 2021. Individuals diagnosed with and/or treated for diabetes were excluded from the study. The body mass index (BMI); plasma concentrations of total, high-density, and low-density lipoprotein cholesterol; triglyceride, glucose, and BCAA; and glycosylated hemoglobin ratio were measured. A basic periodontal examination was performed after the SOB test. Results: The median age of participants (men—20; women—44) was 55 (range, 41–78) years. The plasma BCAA concentration in the SOB-positive group (477 [400–658] μmol/L) was higher than that in the SOB-negative group (432 [307–665] μmol/L). Linear regression analysis revealed that SOB remained independently associated with the plasma BCAA level with statistical significance (β = 0.17, p = 0.02) after adjusting for sex, age, and BMI. Conclusions: SOB was positively correlated with plasma BCAA levels in middle-aged Japanese individuals. Thus, SOB may be a predictor of elevated plasma BCAA levels.

Positive association of branched-chain amino acids with triglyceride and glycated haemoglobin in Indian patients with type 2 diabetes mellitus

Background and aimsOver the past few years, branched-chain amino acids (BCAAs) are increasingly being linked to insulin resistance and type 2 diabetes mellitus (T2DM), but their relevance for metabolic dyslipidaemia in T2DM is unclear. This study aims to determine the plasma and urinary BCAAs and their association with insulin resistance, lipid profile and glycated haemoglobin in patients with T2DM among Indian adults. MethodsIn this analytical cross-sectional study, a total of eighty subjects were recruited, 40 T2DM cases and 40 healthy controls. Blood samples collected were subjected to fasting blood sugar (FBS), lipid profile, HbA1c, insulin and BCAAs analysis and urine samples were assessed for BCAAs. All associations were assessed using Spearman Rank Correlation. ResultsThe plasma levels of BCAAs were significantly higher (p < 0.05) in subjects with T2DM than in control subjects. Spearman Rank Correlation analyses revealed a non-significant (p = 0.21) but positive association between BCAAs and homeostasis model assessment of insulin resistance (HOMA-IR) in patients with T2DM (Rho: 0.27). Among lipid profile parameters, only triglycerides had a significant positive correlation to plasma BCAAs in cases (Rho: 0.5971) but not in control subjects. Findings also revealed a significant positive (p < 0.05) association between plasma BCAAs and HbA1c in patients with T2DM (Rho: 0.5325). Urinary BCAAs levels had a non-significant increase in T2DM subjects and did not show any significant correlation with other parameters assessed. ConclusionElevated levels of plasma BCAAs are positively associated with triglyceride and HbA1c. They could serve as an effective marker for the assessment of metabolic dyslipidaemia in subjects with T2DM. Further, large scale studies are needed for confirmation of the same.

Abstract A45: Diet-induced obesity and caloric restriction weight loss in Diversity Outbred (DO) mice: An experimental preclinical translational model for the investigation of pathways for prevention of obesity and cancer

Abstract Obesity has reached epidemic levels worldwide. It is associated with major chronic diseases, including cancer and cardiovascular disease. The etiology of obesity-associated cancer is poorly understood. New population-based research mouse models are required to better understand prevention and to increase the translational potential of mouse models to humans. The Collaborative Cross (CC) recombinant inbred lines of mice are a population-based model created from an 8-way multiparental advanced generation intercross with a 12% minor allele frequency (MAF) and more than 45 million SNP and CNV, including a significant number of private (unique) alleles. The Diversity Outbred (DO) mice were created from early CC incipient lines (generations G2:F2-5) by random selection and 175 randomly selected breeding pairs. The genetic diversity of the DO and CC RIL mice is ideal for forward and reverse genetics approaches, respectively, to identify potential causally related risk variants and identify etiology based upon causal relationships. Here, we present data from two cohorts (from different breeding generations) of 50 female and 50 male DO mice each (total 100 each sex). After 12-wks high-fat (60% kcal fat) diet we observed significant differences in weight gain during diet-induced obesity DIO. After switching diets for 8-wks of 30% caloric restriction of a low-fat (10% kcal fat) diet, we observed significant interindividual differences in weight loss. Body weight, lean and fat mass differences), increases in 4-hr fasted blood glucose and insulin levels, insulin resistance (HOMA-IR and increased blood branched chain amino acids levels (BCAA) were observed. Four interacting quantitative trait loci/gene (Chst10, Bmiq5, Ath8, and Tcq14) related to metabolism, body mass index, cardiovascular disease, and total cholesterol levels, respectively, have been identified. Regression analysis suggests significant interactions between fasted blood glucose and fat mass, HOMA-IR and fat mass, and BCAA and fat mass in DIO and CR outcomes. Results to date show significant amounts of interindividual variability of each of the quantified phenotypes in response to both diet interventions were demonstrated in female and male DO mice. These data suggest potential avenues of research on obesity and cancer relationships and pathways for intervention. Citation Format: Melissa VerHague, Jody Albright, Salvador Favela, Kerri Barron, Michael F. Coleman, Katie Meyer, John E. French, Stephen D. Hursting. Diet-induced obesity and caloric restriction weight loss in Diversity Outbred (DO) mice: An experimental preclinical translational model for the investigation of pathways for prevention of obesity and cancer [abstract]. In: Proceedings of the AACR Special Conference on Environmental Carcinogenesis: Potential Pathway to Cancer Prevention; 2019 Jun 22-24; Charlotte, NC. Philadelphia (PA): AACR; Can Prev Res 2020;13(7 Suppl): Abstract nr A45.

Impaired Skeletal Muscle Branched-Chain Amino Acids Catabolism Contributes to Their Increased Circulating Levels in a Non-Obese Insulin-Resistant Fructose-Fed Rat Model.

Elevated plasma branched-chain amino acids (BCAA) levels are often observed in obese insulin-resistant (IR) subjects and laboratory animals. A reduced capacity of the adipose tissues (AT) to catabolize BCAA has been proposed as an explanation, but it seems restricted to obesity models of genetically modified or high fat–fed rodents. We aimed to determine if plasma BCAA levels were increased in a model of IR without obesity and to explore the underlying mechanisms. Rats were fed with a standard diet, containing either starch or fructose. BCAA levels, body weight and composition were recorded before and after 5, 12, 30, or 45 days of feeding. Elevated blood BCAA levels were observed in our IR model with unaltered body weight and composition. No changes were observed in the liver or the AT, but instead an impaired capacity of the skeletal muscle to catabolize BCAA was observed, including reduced capacity for transamination and oxidative deamination. Although the elevated blood BCAA levels in the fructose-fed rat seem to be a common feature of the IR phenotype observed in obese subjects and high fat–fed animals, the mechanisms involved in such a metabolic phenomenon are different, likely involving the skeletal muscle BCAA metabolism.

Time-course changes in circulating branched-chain amino acid levels and metabolism in obese Yucatan minipig

High-fat high-sucrose diet (HFHS) overfeeding is one of the main factors responsible for the increased prevalence of metabolic disorders. Elevated levels of branched-chain amino acids (BCAAs) have been associated with metabolic dysfunctions, including insulin resistance (IR). The aim of this study was to elucidate whether elevated BCAA levels are the cause or the consequence of IR and to determine the mechanisms and tissues involved in such a phenotype. We performed a 2-mo follow-up on minipigs overfed an HFHS diet and focused on kinetics fasting and postprandial (PP) BCAA levels and BCAA catabolism in key tissues. The study of the fasting BCAA elevation reveals that BCAA accumulation in the plasma compartment is well correlated with IR markers and body weight. Furthermore, the PP excursion of BCAA levels after the last HFHS meal was exacerbated when compared with that of the first meal, suggesting a reduced amino acid oxidation potential. Although only minor changes in BCAA metabolism were observed in liver, muscle, and the visceral adipose tissue, the oxidative deamination potential of the subcutaneous adipose tissue was blunted after 60 d of HFHS feeding. To our knowledge, the present results demonstrated for the first time in a swine model of obesity and IR, the existence of a phenotype related to high-circulating BCAA levels and metabolic dysregulation. The oxidative BCAA capacity reduction specifically in the subcutaneous adipose tissue emerges, at least in the present swine model, as the more plausible metabolic explanation for the elevated blood BCAA phenotype.

Abstract 104: Defective Branched-chain Amino Acid Catabolism Impairs Glucose Metabolism and Sensitizes the Heart to Ischemia/Reperfusion Injury

The branched-chain amino acids (BCAA) are essential amino acids required for protein homeostasis, energy balance, and nutrient signaling. It has been shown recently that elevations in the circulating BCAAs and related metabolites are associated with insulin resistance and ischemic heart disease, suggesting an important link between BCAA metabolism and cardiac pathology. BCAA catabolism in mitochondria is regulated by the branched-chain keto acid dehydrogenase complex (BCKDC). A mitochondria localized phosphatase 2C (PP2Cm) has been identified to activate BCKDC, serving as a key regulator of BCAA catabolism. Deletion of PP2Cm (KO) increased blood BCAA levels but did not alter cardiac function in unstressed mice. Using 13 C- and 31 P-NMR spectroscopy, we found a significant reduction in the contribution of glucose to oxidative metabolism in KO hearts (16±3 vs. 26±2% for KO and wild-type (WT), respectively, P &lt;0.05), which was accompanied by an ~25% reduction in glucose uptake rate ( P &lt;0.05). Cardiac glycogen content was also reduced (4.4±0.5 vs. 10.9±1.8 μmol glucose/g, P &lt;0.05). In isolated mitochondria from KO or WT hearts treated with BCAAs, significant reductions in pyruvate supported respiration were observed. In enzyme kinetics experiment, acute exposure to BCAAs inhibited pyruvate dehydrogenase complex (PDH) activity in a dose-dependent manner. In isolated perfused hearts subjected to ischemia/reperfusion (I/R), KO hearts showed a greater increase of diastolic pressure during ischemia ( P &lt;0.05), which persisted throughout the reperfusion ( P &lt;0.05). The contractile function in KO barely recovered while it recovered to ~40% of baseline in WT at the end of reperfusion. Increasing glucose supply via overexpression of GLUT1, an insulin-independent glucose transporter, fully restored the impaired recovery to I/R in KO mice. These results show that BCAA catabolism deficiency, as a result of PP2Cm deletion, disrupts normal glucose metabolism and renders the heart highly sensitive to acute ischemia/reperfusion (I/R) injury. These findings reveal an important role of BCAA catabolism in regulating glucose metabolism and cardiac stress response.

Clinical, biochemical and genetic features of 13 children with maple syrup urine disease

Objective To explore the clinical, treatment, and genetic findings of maple syrup urine disease (MSUD). Methods Six boys and 7 girls out of 13 patients from unrelated Chinese families were enrolled.MSUD was detected in one patient by newborn screening.Twelve patients had the onset from the age of 2 days to 1 year and 6 months.They presented vomiting, feeding difficulties, lethargy, coma and psychomotor retardation.Blood amino acids were analyzed, and gene analysis was performed. Results The patients were hospitalized, age ranging from 16 days to 1 year and 8 months.Among them, 11 cases were of classical type and had very early onset.Only 1 patient with interme-diate type had relatively late onset.The girl detected by newborn screening was treated at the age of 9 days.She was healthy now with unknown MSUD type.Eleven classical MSUD children were diagnosed because of significantly elevated blood branched chain aminoacids (leucine, isoleucine, valine and alloisoleucine). Fourteen mutations were found from 10 patients who accepted gene analysis, including 7 mutations in BCKDHA gene (c.178G>T, c. 491T>C, c. 659C>T, c. 740A>G, c. 1214_1219dupCCAACC, c.1234G>A and IVS6+ 1delG0), 5 mutations in BCKDHB gene (c.482T>G, c. 508C>T, c. 767A>G, c. 768C>G and IVS4, -2A>C), 2 mutations in DBT gene (c.1A>G and c. 1150A>G). Two patients had normal development.One patient died from respiratory failure at the age of 4 years and 6 months. Conclusions MSUD is a rare life-threatening disorder which leads to metabolic crisis and brain damage with high mortality.Neonatal screening, early diagnosis and dietary treatment are crucial to improve the outcome. Key words: Maple syrup urine disease; Branched chain amino acid; BCKDHA gene; BCKDHB gene; DBT gene

Eleven novel mutations of the BCKDHA, BCKDHB and DBT genes associated with maple syrup urine disease in the Chinese population: Report on eight cases

Maple syrup urine disease (MSUD) is a rare autosomal recessive disorder that affects the degradation of branched chain amino acids (BCAAs). Only a few cases of MSUD have been documented in Mainland China, and prenatal diagnosis has not been performed so far. In this report, 8 patients (4 girls and 4 boys) with MSUD from 8 unrelated Chinese families were diagnosed at the age of 9 days to 1 year and 8 months. The diagnosis was confirmed by serum BCAAs and genetic analyses. Among the 8 patients, only one was detected by newborn screening. The remaining 7 patients were admitted because of neurological disorders and underwent selective screening. Significantly elevated BCAAs were observed in 7 patients. One patient was diagnosed by post-mortem study. 12 mutations were found in the BCKDHA, BCKDHB and DBT genes. 11 of these mutations were novel: c.178G > T, c.491T > C, c.740A > G, c.1214_1219dupCCAACC and IVS6+1delG in BCKDHA; c.482T > G, c.508C > T, c.767A > G, c.768C > G and IVS4,−2A > C in BCKDHB; and c.1A > G in DBT. Only one mutation, c.659C > T in the BCKDHA gene, had been previously reported. 7 patients were treated by dietary intervention and symptomatic therapy. 6 of them showed clinical improvement. The mother of one patient who died from MSUD underwent amniocentesis during her second pregnancy. The BCAAs level in her amniotic fluid was normal. Only one heterozygous mutation, IVS4,−2A > C in the BCKDHB gene, was detected in the cultured amniocytes. The results revealed that the fetus was not affected by MSUD. Normal development and the blood BCAAs profile confirmed the prenatal diagnosis after birth. Thus, we identified eleven novel mutations associated with MSUD in the Chinese population. Prenatal diagnosis of MSUD was successfully performed on one fetus by genetic analysis of the cultured amniocytes.

Brain-blood amino acid correlates following protein restriction in murine maple syrup urine disease.

BackgroundConventional therapy for patients with maple syrup urine disease (MSUD) entails restriction of protein intake to maintain acceptable levels of the branched chain amino acid, leucine (LEU), monitored in blood. However, no data exists on the correlation between brain and blood LEU with protein restriction, and whether correction in blood is reflected in brain.MethodsTo address this question, we fed intermediate MSUD mice diets of 19% (standard) and 6% protein, with collection of sera (SE), striata (STR), cerebellum (CE) and cortex (CTX) for quantitative amino acid analyses.ResultsLEU and valine (VAL) levels in all brain regions improved on average 28% when shifting from 19% to 6% protein, whereas the same improvements in SE were on average 60%. Isoleucine (ILE) in brain regions did not improve, while the SE level improved 24% with low-protein consumption. Blood-branched chain amino acids (LEU, ILE, and VAL in sera (SE)) were 362-434 μM, consistent with human values considered within control. Nonetheless, numerous amino acids in brain regions remained abnormal despite protein restriction, including glutamine (GLN), aspartate (ASP), glutamate (GLU), gamma-aminobutyric acid (GABA), asparagine (ASN), citrulline (CIT) and serine (SER). To assess the specificity of these anomalies, we piloted preliminary studies in hyperphenylalaninemic mice, modeling another large neutral aminoacidopathy. Employing an identical dietary regimen, we found remarkably consistent abnormalities in GLN, ASP, and GLU.ConclusionsOur results suggest that blood amino acid analysis may be a poor surrogate for assessing the outcomes of protein restriction in the large neutral amino acidopathies, and further indicate that chronic neurotransmitter disruptions (GLU, GABA, ASP) may contribute to long-term neurocognitive dysfunction in these disorders.

Branched-chain Amino Acid Intake and the Risk of Diabetes in a Japanese Community: The Takayama Study

Dietary supplementation with branched-chain amino acids (BCAAs), including leucine, isoleucine, and valine, has shown potential benefits for the metabolic profile. However, higher blood BCAA levels have been associated with insulin resistance. To our knowledge, there has been no study on dietary BCAAs and the risk of diabetes. We examined the association between BCAA intake and risk of diabetes in a population-based cohort study in Japan. A total of 13,525 residents of Takayama City, Japan, who enrolled in a cohort study in 1992 responded to a follow-up questionnaire seeking information about diabetes in 2002. Diet at baseline was assessed by means of a validated food frequency questionnaire. A high intake of BCAAs in terms of percentage of total protein was significantly associated with a decreased risk of diabetes in women after controlling for covariates; the hazard ratio for the highest tertile versus the lowest was 0.57 (95% confidence interval: 0.36, 0.90; P-trend = 0.02). In men, leucine intake was significantly marginally associated with the risk of diabetes; the hazard ratio for the highest tertile versus the lowest was 0.70 (95% confidence interval: 0.48, 1.02; P-trend = 0.06). Data suggest that a high intake of BCAAs may be associated with a decrease in the risk of diabetes.

Regulation of adipose branched-chain amino acid catabolism enzyme expression and cross-adipose amino acid flux in human obesity

Elevated blood branched-chain amino acids (BCAA) are often associated with insulin resistance and type 2 diabetes, which might result from a reduced cellular utilization and/or incomplete BCAA oxidation. White adipose tissue (WAT) has become appreciated as a potential player in whole body BCAA metabolism. We tested if expression of the mitochondrial BCAA oxidation checkpoint, branched-chain α-ketoacid dehydrogenase (BCKD) complex, is reduced in obese WAT and regulated by metabolic signals. WAT BCKD protein (E1α subunit) was significantly reduced by 35-50% in various obesity models (fa/fa rats, db/db mice, diet-induced obese mice), and BCKD component transcripts significantly lower in subcutaneous (SC) adipocytes from obese vs. lean Pima Indians. Treatment of 3T3-L1 adipocytes or mice with peroxisome proliferator-activated receptor-γ agonists increased WAT BCAA catabolism enzyme mRNAs, whereas the nonmetabolizable glucose analog 2-deoxy-d-glucose had the opposite effect. The results support the hypothesis that suboptimal insulin action and/or perturbed metabolic signals in WAT, as would be seen with insulin resistance/type 2 diabetes, could impair WAT BCAA utilization. However, cross-tissue flux studies comparing lean vs. insulin-sensitive or insulin-resistant obese subjects revealed an unexpected negligible uptake of BCAA from human abdominal SC WAT. This suggests that SC WAT may not be an important contributor to blood BCAA phenotypes associated with insulin resistance in the overnight-fasted state. mRNA abundances for BCAA catabolic enzymes were markedly reduced in omental (but not SC) WAT of obese persons with metabolic syndrome compared with weight-matched healthy obese subjects, raising the possibility that visceral WAT contributes to the BCAA metabolic phenotype of metabolically compromised individuals.

Large neutral amino acids: dietary effects on brain neurochemistry and function

The ingestion of large neutral amino acids (LNAA), notably tryptophan, tyrosine and the branched-chain amino acids (BCAA), modifies tryptophan and tyrosine uptake into brain and their conversion to serotonin and catecholamines, respectively. The particular effect reflects the competitive nature of the transporter for LNAA at the blood-brain barrier. For example, raising blood tryptophan or tyrosine levels raises their uptake into brain, while raising blood BCAA levels lowers tryptophan and tyrosine uptake; serotonin and catecholamine synthesis in brain parallel the tryptophan and tyrosine changes. By changing blood LNAA levels, the ingestion of particular proteins causes surprisingly large variations in brain tryptophan uptake and serotonin synthesis, with minimal effects on tyrosine uptake and catecholamine synthesis. Such variations elicit predictable effects on mood, cognition and hormone secretion (prolactin, cortisol). The ingestion of mixtures of LNAA, particularly BCAA, lowers brain tryptophan uptake and serotonin synthesis. Though argued to improve physical performance by reducing serotonin function, such effects are generally considered modest at best. However, BCAA ingestion also lowers tyrosine uptake, and dopamine synthesis in brain. Increasing dopamine function in brain improves performance, suggesting that BCAA may fail to increase performance because dopamine is reduced. Conceivably, BCAA administered with tyrosine could prevent the decline in dopamine, while still eliciting a drop in serotonin. Such an LNAA mixture might thus prove an effective enhancer of physical performance. The thoughtful development and application of dietary proteins and LNAA mixtures may thus produce treatments with predictable and useful functional effects.

Markers Associated with Inborn Errors of Metabolism of Branched-Chain Amino Acids and Their Relevance to Upper Levels of Intake in Healthy People: An Implication from Clinical and Molecular Investigations on Maple Syrup Urine Disease

Maple syrup urine disease (MSUD) is caused by a deficiency in the branched-chain alpha-ketoacid dehydrogenase complex. Accumulations of branched-chain amino acids (BCAAs) and branched-chain alpha-ketoacids (BCKAs) in patients with MSUD induce ketoacidosis, neurological disorders, and developmental disturbance. BCAAs and BCKAs influence on the nervous system can be estimated by analyzing these patients. According to clinical investigations on MSUD patients, leucine levels over 400 micromol/L apparently can cause any clinical problem derived from impaired function of the central nervous system. Damage to neuronal cells found in MSUD patients are presumably because of higher concentrations of both blood BCAAs or BCKAs, especially alpha-ketoisocapronic acids. These clinical data from MSUD patients provide a valuable basis on understanding leucine toxicity in the normal subject.

ENU mutagenesis identifies mice with mitochondrial branched-chain aminotransferase deficiency resembling human maple syrup urine disease

Tandem mass spectrometry was applied to detect derangements in the pathways of amino acid and fatty acid metabolism in N-ethyl-N-nitrosourea-treated (ENU-treated) mice. We identified mice with marked elevation of blood branched-chain amino acids (BCAAs), ketoaciduria, and clinical features resembling human maple syrup urine disease (MSUD), a severe genetic metabolic disorder caused by the deficiency of branched-chain alpha-keto acid dehydrogenase (BCKD) complex. However, the BCKD genes and enzyme activity were normal. Sequencing of branched-chain aminotransferase genes (Bcat) showed no mutation in the cytoplasmic isoform (Bcat-1) but revealed a homozygous splice site mutation in the mitochondrial isoform (Bcat-2). The mutation caused a deletion of exon 2, a marked decrease in Bcat-2 mRNA, and a deficiency in both BCAT-2 protein and its enzyme activity. Affected mice responded to a BCAA-restricted diet with amelioration of the clinical symptoms and normalization of the amino acid pattern. We conclude that BCAT-2 deficiency in the mouse can cause a disease that mimics human MSUD. These mice provide an important animal model for study of BCAA metabolism and its toxicity. Metabolomics-guided screening, coupled with ENU mutagenesis, is a powerful approach in uncovering novel enzyme deficiencies and recognizing important pathways of genetic metabolic disorders.

Changes in brain ECF amino acids in rats with experimentally induced hyperammonemia.

Using microdialysis, we studied brain extracellular fluid (ECF) amino acid metabolism in rats with experimentally induced hyperammonemia and regional elevation of brain ECF ammonia levels. The total brain ECF amino acid level was increased by an elevation of the blood ammonia level. Hyperammonemia elevated brain ECF aromatic amino acids and reduced arterial blood branched chain amino acids. When rats with hyperammonemia were intravenously administered norleucine, the brain ECF norleucine level rose markedly, suggesting increased permeability of the blood-brain barrier. When rats with hyperammonemia were infused with a branched chain amino acid-rich preparation, the elevated brain ECF aromatic amino acids level was not suppressed. Following local intracerebral ammonia infusion, only glutamate levels showed a marked elevation. These results suggest that impairment of the blood-brain barrier related to hyperammonemia increases the inflow of low molecular weight substances including amino acids. Furthermore, the ammonia-induced increase of glutamate in the cerebral ECF suggests that high ammonia levels may increase the excitability of the brain. Thus, ammonia may serve as a key factor in the onset of hepatic encephalopathy.

Valine metabolism in vivo: Effects of high dietary levels of leucine and isoleucine

The short-term effects of feeding rats high levels of L-leucine or L-isoleucine on valine metabolism in vivo have been investigated. Consumption of a low-protein diet containing an additional 5% of leucine resulted in depression within one hour of the plasma concentrations of isoleucine, valine, α-keto-β-methylvalerate, and α-ketoisovalerate. Concurrently with these changes in blood branched-chain amino acids and branched-chain ketoacids was a rapid increase (51%) in whole-body L-[1- 14C]-valine oxidation. Studies with intragastrically administered leucine solutions indicated that the depressions in blood concentrations of valine occurred over the same time period as the stimulation in valine oxidation. In contrast, consumption of a low-protein diet containing an additional 5% of isoleucine had no significant effect on the plasma concentrations of leucine, valine, and α-ketoisocaproate; a significant ( P < 0.01) depression in the plasma concentration of α-ketoisovalerate was observed three hours after the diet containing excess isoleucine had been consumed. In contrast to the results obtained with excess leucine, consumption of excess isoleucine had no significant effect on the rate of valine oxidation in vivo. As part of an effort to explain the leucine-induced depletion of plasma valine and stimulation of valine oxidation, liver and muscle branched-chain aminotransferase and liver branched-chain ketoacid dehydrogenase activities were measured. Consumption of excess leucine had no significant effect on either muscle or liver aminotransferase activities, but was associated with a greater than two-fold increase in hepatic dehydrogenase activity. Also, consumption of excess leucine resulted in an increase in the percentage of active dehydrogenase from 43 ± 5% to 80 ± 4%.