BDK knockout skeletal muscle satellite cells exhibit enhanced protein translation initiation signal in response to BCAA in vitro.

We examined the effects of branched-chain amino acids (BCAAs) and electrical pulse stimulation (EPS) on the mTORC1 pathway in muscle satellite cells (MSCs) isolated from branched-chain α-keto acid dehydrogenase kinase (BDK) knockout (KO) mice in vitro. MSCs were isolated from BDK KO and wild-type (WT) mice, proliferated, and differentiated into myotubes. BCAA stimulation increased the phosphorylation of p70 S6 kinase (p70S6K), a marker of protein translation initiation, in MSCs from WT and BDK KO mice, but the rate of the increase was higher in MSCs isolated from BDK KO mice. Contrarily, there was no difference in the increase in p70S6K phosphorylation by EPS. Acute BDK knockdown in MSCs from WT mice using shRNA decreased p70S6K phosphorylation in response to BCAA stimulation. Collectively, the susceptibility of mTORC1 to BCAA stimulation was elevated by chronic, but not acute, enhancement of BCAA catabolism.

Negative regulation of amino acid signaling by MAPK-regulated 4F2hc/Girdin complex.

Amino acid signaling mediated by the activation of mechanistic target of rapamycin complex 1 (mTORC1) is fundamental to cell growth and metabolism. However, how cells negatively regulate amino acid signaling remains largely unknown. Here, we show that interaction between 4F2 heavy chain (4F2hc), a subunit of multiple amino acid transporters, and the multifunctional hub protein girders of actin filaments (Girdin) down-regulates mTORC1 activity. 4F2hc interacts with Girdin in mitogen-activated protein kinase (MAPK)- and amino acid signaling-dependent manners to translocate to the lysosome. The resultant decrease in cell surface 4F2hc leads to lowered cytoplasmic glutamine (Gln) and leucine (Leu) content, which down-regulates amino acid signaling. Consistently, Girdin depletion augments amino acid-induced mTORC1 activation and inhibits amino acid deprivation-induced autophagy. These findings uncovered the mechanism underlying negative regulation of amino acid signaling, which may play a role in tightly regulated cell growth and metabolism.

Antihypertensive drug valsartan as a novel BDK inhibitor.

Catabolism of branched-chain amino acids (BCAAs) is affected by various physiological conditions and its abnormality is associated with glucose metabolism, heart disease, and neurological dysfunction. The first two steps of the BCAA metabolic pathway are common to the three BCAAs (leucine, isoleucine, and valine). The second step is an irreversible rate-limited reaction catalyzed by branched-chain α-keto acid dehydrogenase (BCKDH), which is bound to a specific kinase, BCKDH kinase (BDK), and inactivated by phosphorylation. Here, we investigated potential new BDK inhibitors and discovered valsartan, an angiotensin II type 1 receptor (AT1R) blocker, as a new BDK inhibitor. BCKDH phosphorylation and the BCKDH-BDK interaction were inhibited by valsartan in vitro. Valsartan administration in rats resulted in increased BCKDH activity by decreasing the dephosphorylated level of BCKDH complex, bound forms of BDK from BCKDH complex as well as decreased plasma BCAA concentrations. Valsartan is a novel BDK inhibitor that competes with ATP, via a different mechanism from allosteric inhibitors. The BDK inhibitor has been shown to preserve cardiac function in pressure overload-induced heart failure mice and to attenuate insulin resistance in obese mice. Our findings suggest that valsartan is a potent seed compound for developing a powerful BDK inhibitor and useful medication for treating heart failure and metabolic diseases with suppressed BCAA catabolism.

Branched-chain amino acids regulate hyaluronan synthesis and PPARα expression in the skin.

We examined the effects of deletion of branched-chain α-keto acid dehydrogenase kinase (BDK), a key enzyme in branched-chain amino acid catabolism, on hyaluronan synthesis in mice. The skin levels of hyaluronan and the gene expression levels of hyaluronan synthase (Has)2, Has3, and peroxisome proliferator-activated receptor-α were significantly lower in the BDK-knockout group than in the wild-type group.

Enhanced Echo Intensity of Skeletal Muscle Is Associated With Exercise Intolerance in Patients With Heart Failure.

BACKGROUND: Skeletal muscle is quantitatively and qualitatively impaired in patients with heart failure (HF), which is closely linked to lowered exercise capacity. Ultrasonography (US) for skeletal muscle has emerged as a useful, noninvasive tool to evaluate muscle quality and quantity. Here we investigated whether muscle quality based on US-derived echo intensity (EI) is associated with exercise capacity in patients with HF. METHODS AND RESULTS: Fifty-eight patients with HF (61 ± 12 years) and 28 control subjects (58 ± 14 years) were studied. The quadriceps femoris echo intensity (QEI) was significantly higher and the quadriceps femoris muscle thickness (QMT) was significantly lower in the patients with HF than the controls (88.3 ± 13.4 vs 81.1 ± 7.5, P= .010; 5.21 ± 1.10 vs 6.54 ±1.34 cm, P< .001, respectively). By univariate analysis, QEI was significantly correlated with age, peak oxygen uptake (VO2), and New York Heart Association class in the HF group. A multivariable analysis revealed that the QEI was independently associated with peak VO2 after adjustment for age, gender, body mass index, and QMT: ß-coefficient = -11.80, 95%CI (-20.73, -2.86), P= .011. CONCLUSION: Enhanced EI in skeletal muscle was independently associated with lowered exercise capacity in HF. The measurement of EI is low-cost, easily accessible, and suitable for assessment of HF-related alterations in skeletal muscle quality.

Ca2+-dependent inhibition of branched-chain α-ketoacid dehydrogenase kinase by thiamine pyrophosphate.

Catabolism of the branched-chain amino acids (BCAAs: leucine, isoleucine, and valine) is regulated by the branched-chain α-ketoacid dehydrogenase (BCKDH) complex, which in turn is regulated by phosphorylation catalyzed by BCKDH kinase (BDK). Thiamine pyrophosphate (TPP) is required as a coenzyme for the E1 component of the BCKDH complex and can also bring about activation of the complex by inhibiting BDK. The present study shows that free Ca2+ in the physiological range greatly increases the sensitivity of BDK to inhibition by TPP (IC50 of 2.5 µM in the presence of 1 µM free Ca2+). This novel mechanism may be responsible for the stimulation of BCAA oxidation by conditions that increase mitochondrial free Ca2+ levels, e.g. in skeletal muscle during exercise.

Physiological and pathological roles of branched-chain amino acids in the regulation of protein and energy metabolism and neurological functions.

Branched-chain amino acids (BCAAs: leucine, isoleucine, and valine) are essential amino acids for humans and play an important role as the building blocks of proteins. Recent studies have disclosed that free BCAAs in the tissue amino acid pool function not only as substrates for protein synthesis, but also as regulators of protein and energy metabolism. Furthermore, BCAAs are actively used as an amino group donor to synthesize glutamate in the brain. These functions of BCAAs are closely related to human health. This review summarizes the recent findings concerning physiological and pathological roles of free BCAAs in the metabolism and neurological functions.

Branched-chain amino acid (BCAA) supplementation enhances adaptability to exercise training of mice with a muscle-specific defect in the control of BCAA catabolism.

Branched-chain α-keto acid dehydrogenase (BCKDH) kinase (BDK) suppresses the branched-chain amino acid (BCAA) catabolism by inactivation of the BCKDH complex. The muscle-specific BDK-deficient (BDK-mKO) mice showed accelerated BCAA oxidation in muscle and decreased endurance capacity after training (Xu et al. PLoS One. 12 (2017) e0180989). We here report that BCAA supplementation overcompensated endurance capacity in BDK-mKO mice after training.

Branched-chain amino acids regulate type I tropocollagen and type III tropocollagen syntheses via modulation of mTOR in the skin.

Branched-chain amino acids (BCAAs) exhibit many physiological functions. However, the potential link and mechanism between BCAA and skin function are unknown. We examined the effects of deletion of branched-chain α-keto acid dehydrogenase kinase (BDK), a key enzyme in BCAA catabolism, on type I and III tropocollagen syntheses in mice. Leucine and isoleucine levels were significantly lower in the skin of BDK-KO mice compared with wild-type mice. No changes in valine concentrations were observed. The levels of type I and III tropocollagen proteins and mRNAs (COL1A1 and COL3A1) were significantly lower in the skin of BDK-KO mice compared with wild-type mice. The phosphorylation of p70 S6 kinase, which indicates mammalian target of rapamycin (mTOR) activation, was reduced in the skin of BDK-KO mice compared with wild-type mice. These findings suggest that deficiencies of leucine and isoleucine reduce type I and III tropocollagen syntheses in skin by suppressing the action of mTOR.

Clinical, biochemical and metabolic characterisation of a mild form of human short-chain enoyl-CoA hydratase deficiency: significance of increased N-acetyl-S-(2-carboxypropyl)cysteine excretion.

BACKGROUND: Short-chain enoyl-CoA hydratase-ECHS1-catalyses many metabolic pathways, including mitochondrial short-chain fatty acid ß-oxidation and branched-chain amino acid catabolic pathways; however, the metabolic products essential for the diagnosis of ECHS1 deficiency have not yet been determined. The objective of this report is to characterise ECHS1 and a mild form of its deficiency biochemically, and to determine the candidate metabolic product that can be efficiently used for neonatal diagnosis. METHODS: We conducted a detailed clinical, molecular genetics, biochemical and metabolic analysis of sibling patients with ECHS1 deficiency. Moreover, we purified human ECHS1, and determined the substrate specificity of ECHS1 for five substrates via different metabolic pathways. RESULTS: Human ECHS1 catalyses the hydration of five substrates via different metabolic pathways, with the highest specificity for crotonyl-CoA and the lowest specificity for tiglyl-CoA. The patients had relatively high (∼7%) residual ECHS1 enzyme activity for crotonyl-CoA and methacrylyl-CoA caused by the compound heterozygous mutations (c.176A>G, (p.N59S) and c.413C>T, (p.A138V)) with normal mitochondrial complex I-IV activities. Affected patients excrete large amounts of N-acetyl-S-(2-carboxypropyl)cysteine, a metabolite of methacrylyl-CoA. CONCLUSIONS: Laboratory data and clinical features demonstrated that the patients have a mild form of ECHS1 deficiency harbouring defective valine catabolic and ß-oxidation pathways. N-Acetyl-S-(2-carboxypropyl) cysteine level was markedly high in the urine of the patients, and therefore, N-acetyl-S-(2-carboxypropyl)cysteine was regarded as a candidate metabolite for the diagnosis of ECHS1 deficiency. This metabolite is not part of current routine metabolic screening protocols, and its inclusion, therefore, holds immense potential in accurate diagnosis.

Regulation of the plasma amino acid profile by leucine via the system L amino acid transporter.

Plasma concentrations of amino acids reflect the intracellular amino acid pool in mammals. However, the regulatory mechanism requires clarification. In this study, we examined the effect of leucine administration on plasma amino acid profiles in mice with and without the treatment of 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH) or rapamycin as an inhibitor of system L or mammalian target of rapamycin complex 1, respectively. The elevation of plasma leucine concentration after leucine administration was associated with a significant decrease in the plasma concentrations of isoleucine, valine, methionine, phenylalanine, and tyrosine; BCH treatment almost completely blocked the leucine-induced decrease in plasma amino acid concentrations. Rapamycin treatment had much less effects on the actions of leucine than BCH treatment. These results suggest that leucine regulates the plasma concentrations of branched-chain amino acids, methionine, phenylalanine, and tyrosine, and that system L amino acid transporters are involved in the leucine action.

Multispecificity of immunoglobulin M antibodies raised against advanced glycation end products: involvement of electronegative potential of antigens.

BACKGROUND: Advanced glycation end products (AGEs) can act as neoantigens to trigger immune responses. RESULTS: Natural IgM antibodies against AGEs recognize multiple molecules, including DNA and chemically modified proteins. CONCLUSION: There is a close relationship between the formation of AGEs and innate immune responses. SIGNIFICANCE: Our findings highlight AGEs and related modified proteins as a source of multispecific natural antibodies Advanced glycation end products (AGEs) are a heterogeneous and complex group of compounds that are formed when reducing sugars, such as dehydroascorbic acid, react in a nonenzymatic way with amino acids in proteins and other macromolecules. AGEs are prevalent in the diabetic vasculature and contribute to the development of atherosclerosis. The presence and accumulation of AGEs in many different cell types affect the extracellular and intracellular structure and function. In the present study, we studied the immune response to the dehydroascorbic acid-derived AGEs and provide multiple lines of evidence suggesting that the AGEs could be an endogenous source of innate epitopes recognized by natural IgM antibodies. Prominent IgM titers to the AGEs were detected in the sera of normal mice and were significantly accelerated by the immunization with the AGEs. Patients with systemic lupus erythematosus (SLE), a potentially fatal systemic autoimmune disease characterized by the increased production of autoantibodies, showed significantly higher serum levels of the IgM titer against the AGEs than healthy individuals. A progressive increase in the IgM response against the AGEs was also observed in the SLE-prone mice. Strikingly, a subset of monoclonal antibodies, showing a specificity toward the AGEs, prepared from normal mice immunized with the AGEs and from the SLE mice cross-reacted with the double-stranded DNA. Moreover, they also cross-reacted with several other modified proteins, including the acetylated proteins, suggesting that the multiple specificity of the antibodies might be ascribed, at least in part, to the increased electronegative potential of the proteins. These findings suggest that the protein modification by the endogenous carbonyl compounds, generating electronegative proteins, could be a source of multispecific natural antibodies.

Effects of ingesting highly branched cyclic dextrin during endurance exercise on rating of perceived exertion and blood components associated with energy metabolism.

We compared the effect of relatively low doses (15 g) of highly branched cyclic dextrin (HBCD) with that of maltodextrin during endurance exercise on the rating of perceived exertion (RPE) in a crossover, double-blind study of healthy volunteers. The RPE increased during exercise and its increase was significantly less at 30 and 60 min after ingesting HBCD than maltodextrin.

Activation of Hepatic Branched-Chain α-Ketoacid Dehydrogenase Complex by Vitamin D Deficiency in Rats.

Branched-chain α-ketoacid dehydrogenase (BCKDH) complex is a rate-limiting enzyme in branched-chain amino acid catabolism and is subject to inactivation via phosphorylation by BCKDH kinase (BDK). In the present study, we examined the effects of vitamin D-deficiency on hepatic BCKDH and BDK activities in rats. Rats fed a vitamin D-deficient diet long-term showed a slight but significant decrease in plasma Ca concentration, which was associated with an elevation of BCKDH activity and a decrease in BDK activity. These results suggest that vitamin D deficiency promotes BCAA catabolism via BCKDH activation, which resulted from BDK suppression. It is proposed that Ca2+-dependent BDK inhibition by thiamine pyrophosphate may be involved in the BDK suppression.

"Nutrient-Repositioning"-Unexpected Amino Acid Functions.

Repositioning is usually used to indicate drug repositioning, or the finding of new disease applications for existing, approved drugs. Nutrients can be ingested for nutritional as well as therapeutic purposes, acting much the same as drugs. Amino acids are organic compounds that possess both amino and carboxy group functionalities and are best known as building blocks of proteins in living organisms. Recent studies of individual amino acids have revealed them to be functional ingredients of new therapeutics, promoting health in addition to nutrition. Here, we propose "nutrient-repositioning", the discovery of effects different from the existing effects of nutrients. This review summarizes some recent discoveries of unexpected amino acid functions, especially in BCAAs, histidine and serine.

Patients with low muscle mass have characteristic microbiome with low potential for amino acid synthesis in chronic liver disease.

Sarcopenia is thought to be related to the microbiome, but not enough reports in chronic liver disease (CLD) patients. In addition to the differences in microbiome, the role of the microbiome in the gut is also important to be clarified because it has recently been shown that the microbiome may produce branched-chain amino acids (BCAAs) in the body. In this single-center study, sixty-nine CLD patients were divided by skeletal muscle mass index (SMI) into low (L-SMI: n = 25) and normal (N-SMI: n = 44). Microbiome was analyzed from stool samples based on V3-4 region of bacterial 16S rRNA). L-SMI had a lower Firmicutes/Bacteroidetes ratio than N-SMI. At the genus level, Coprobacillus, Catenibacterium and Clostridium were also lower while the Bacteroides was higher. Predictive functional profiling of the L-SMI group showed that genes related to nitrogen metabolism were enriched, but those related to amino acid metabolism, including BCAA biosynthesis, were lower. The genes related to 'LPS biosynthesis' was also higher. The microbiome of CLD patients with low muscle mass is characterized not only by high relative abundance of gram-negative bacteria with LPS, but also by the possibility of low potential for amino acid synthesis including BCAAs.

Effect of branched-chain amino acid supplementation during unloading on regulatory components of protein synthesis in atrophied soleus muscles.

Maintenance of skeletal muscle mass depends on the equilibrium between protein synthesis and protein breakdown; diminished functional demand during unloading breaks this balance and leads to muscle atrophy. The current study analyzed time-course alterations in regulatory genes and proteins in the unloaded soleus muscle and the effects of branched-chain amino acid (BCAA) supplementation on muscle atrophy and abundance of molecules that regulate protein turnover. Short-term (6 days) hindlimb suspension of rats resulted in significant losses of myofibrillar proteins, total RNA, and rRNAs and pronounced atrophy of the soleus muscle. Muscle disuse induced upregulation and increases in the abundance of the eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1), increases in gene and protein amounts of two ubiquitin ligases (muscle RING-finger protein 1 and muscle atrophy F-box protein), and decreases in the expression of cyclin D1, the ribosomal protein S6 kinase 1, the mammalian target of rapamycin (mTOR), and ERK1/2. BCAA addition to the diet did not prevent muscle atrophy and had no apparent effect on regulators of proteasomal protein degradation. However, BCAA supplementation reduced the loss of myofibrillar proteins and RNA, attenuated the increases in 4E-BP1, and partially preserved cyclin D1, mTOR and ERK1 proteins. These results indicate that BCAA supplementation alone does not oppose protein degradation but partly preserves specific signal transduction proteins that act as regulators of protein synthesis and cell growth in the non-weight-bearing soleus muscle.

Urinary l-erythro-ß-hydroxyasparagine-a novel serine racemase inhibitor and substrate of the Zn2+-dependent d-serine dehydratase.

In the present study, we identified l-erythro-ß-hydroxyasparagine (l-ß-EHAsn) found abundantly in human urine, as a novel substrate of Zn2+-dependent d-serine dehydratase (DSD). l-ß-EHAsn is an atypical amino acid present in large amounts in urine but rarely detected in serum or most organs/tissues examined. Quantitative analyses of urinary l-ß-EHAsn in young healthy volunteers revealed significant correlation between urinary l-ß-EHAsn concentration and creatinine level. Further, for in-depth analyses of l-ß-EHAsn, we developed a simple three-step synthetic method using trans-epoxysuccinic acid as the starting substance. In addition, our research revealed a strong inhibitory effect of l-ß-EHAsn on mammalian serine racemase, responsible for producing d-serine, a co-agonist of the N-methyl-d-aspartate (NMDA) receptor involved in glutamatergic neurotransmission.

Magnesium depletion extends fission yeast lifespan via general amino acid control activation.

Nutrients including glucose, nitrogen, sulfur, zinc, and iron are involved in the regulation of chronological lifespan (CLS) of yeast, which serves as a model of the lifespan of differentiated cells of higher organisms. Herein, we show that magnesium (Mg2+ ) depletion extends CLS of the fission yeast Schizosaccharomyces pombe through a mechanism involving the Ecl1 gene family. We discovered that ecl1+ expression, which extends CLS, responds to Mg2+ depletion. Therefore, we investigated the underlying intracellular responses. In amino acid auxotrophic strains, Mg2+ depletion robustly induces ecl1+ expression through the activation of the general amino acid control (GAAC) pathway-the equivalent of the amino acid response of mammals. Polysome analysis indicated that the expression of Ecl1 family genes was required for regulating ribosome amount when cells were starved, suggesting that Ecl1 family gene products control the abundance of ribosomes, which contributes to longevity through the activation of the evolutionarily conserved GAAC pathway. The present study extends our understanding of the cellular response to Mg2+ depletion and its influence on the mechanism controlling longevity.

Supplementation of 1-Kestose Modulates the Gut Microbiota Composition to Ameliorate Glucose Metabolism in Obesity-Prone Hosts.

Insulin resistance leads to the onset of medical conditions such as type 2 diabetes, and its development is associated with the alteration in the gut microbiota. Although it has been demonstrated that supplementation with prebiotics modulates the gut microbiota, limited evidence is available for effects of prebiotics on insulin resistance, especially for humans. We investigated the prebiotic effect of 1-kestose supplementation on fasting insulin concentration in obesity-prone humans and rats. In the preliminary study using rats, the hyperinsulinemia induced by high-fat diet was suppressed by intake of water with 2% (w/v) 1-kestose. In the clinical study using obese-prone volunteers, the fasting serum insulin level was significantly reduced from 6.5 µU/mL (95% CI, 5.5-7.6) to 5.3 (4.6-6.0) by the 12-week intervention with supplementation of 10 g 1-kestose/day, whereas it was not changed by the intervention with placebo (6.2 µU/mL (5.4-7.1) and 6.5 (5.5-7.6) before and after intervention, respectively). The relative abundance of fecal Bifidobacterium was significantly increased to 0.3244 (SD, 0.1526) in 1-kestose-supplemented participants compared to that in control participants (0.1971 (0.1158)). These results suggest that prebiotic intervention using 1-kestose may potentially ameliorate insulin resistance in overweight humans via the modulation of the gut microbiota. UMIN 000028824.