Effects of 1-kestose on microbiome changes caused by vonoprazan: a randomized, double-blind, placebo-controlled pilot study.

BACKGROUND AND AIM: Potassium-competitive acid blockers more strongly suppress the gastric acid barrier than proton pump inhibitors and cause dysbiosis. However, preventive measures in this regard have not been established. We aimed to evaluate whether 1-kestose, a known prebiotic, was effective at alleviating dysbiosis caused by potassium-competitive acid blockers. METHODS: Patients scheduled to undergo endoscopic resection for superficial gastroduodenal tumors were enrolled and randomized 1:1 to receive either 1-kestose or placebo. All patients were started on potassium-competitive acid blocker (vonoprazan 20 mg/day) and took 1-kestose 10 g/day or placebo (maltose) 5 g/day for 8 weeks. The primary outcome was the effect of 1-kestose on potassium-competitive acid blocker-induced alterations in the microbiome. The fecal microbiome was analyzed before and after potassium-competitive acid blocker treatment via MiSeq (16S rRNA gene V3-V4 region). RESULTS: Forty patients were enrolled, and 16 in each group were analyzed. In the placebo group, the Simpson index, an alpha diversity, was significantly decreased and relative abundance of Streptococcus was significantly increased by 1.9-fold. In the kestose group, the Simpson index did not change significantly and relative abundance of Streptococcus increased 1.3-fold, but this was not a significant change. In both groups, no adverse events occurred, ulcers were well healed, and pretreatment and posttreatment short-chain fatty acid levels did not differ. CONCLUSIONS: The potassium-competitive acid blocker caused dysbiosis in the placebo group; this effect was prevented by 1-kestose. Thus, 1-kestose may be useful in dysbiosis treatment.

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.

Tolerable amounts of amino acids for human supplementation: summary and lessons from published peer-reviewed studies.

Amino acid supplementation may be indicated to correct for insufficient amino acid intake in healthy individuals, and in specific physiological or pathophysiological situations. However, there is a concern to not supplement beyond the tolerable upper intake level (UL) by determining parameters of no-observed-adverse-effect level (NOAEL) or lowest-observed-adverse-effect level (LOAEL) for each amino acid. Since the NOAEL and LOAEL values are at least one order of magnitude different when comparing the values obtained in rats and humans, the aim of this review is to evaluate to what extent the amino acid UL measured in the rat model, when referenced to the dietary usual consumption (UC) and dietary requirement (RQ) for indispensable amino acids, may be used as an approximation of the UL in humans. This review then compares the ratios of the NOAEL or LOAEL over UC and RQ in the rat model with the same ratios calculated in humans for the nine amino acids (arginine, serine, glycine, histidine, leucine, lysine, methionine, phenylalanine, and tryptophan) for which this comparison can be done. From the calculations made, it appears that for these 9 amino acids, the calculated ratios for rats and humans, although rather different for several amino acids, remains for all of them in the same order of magnitude. For tryptophan, tyrosine, and valine, the ratios calculated in rats are markedly different according to the sex of animals, raising the view that it may be also the case in humans.

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.

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.

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.

Efficacy of 1-kestose supplementation in patients with mild to moderate ulcerative colitis: A randomised, double-blind, placebo-controlled pilot study.

BACKGROUND: Ulcerative colitis involves an excessive immune response to intestinal bacteria. Whether administering prebiotic 1-kestose is effective for active ulcerative colitis remains controversial. AIMS: This randomised, double-blind, placebo-controlled pilot trial investigated the efficacy of 1-kestose against active ulcerative colitis. METHODS: Forty patients with mild to moderate active ulcerative colitis were randomly treated with 1-kestose (N = 20) or placebo (maltose, N = 20) orally for 8 weeks in addition to the standard treatment. The Lichtiger clinical activity index and Ulcerative Colitis Endoscopic Index of Severity were determined. Faecal samples were analysed to evaluate the gut microbiome and metabolites. RESULTS: The clinical activity index at week 8 was significantly lower in the 1-kestose group than in the placebo group (3.8 ± 2.7 vs. 5.6 ± 2.1, p = 0.026). Clinical remission and response rates were higher in the 1-kestose group than in the placebo group (remission: 55% vs. 20%, p = 0.048; response: 60% vs. 25%, p = 0.054). The Ulcerative Colitis Endoscopic Index of Severity at week 8 was not significantly different (2.8 ± 1.6 vs. 3.5 ± 1.6, p = 0.145). Faecal analysis showed significantly reduced alpha-diversity in the 1-kestose group, with a decreased relative abundance of several bacteria, including Ruminococcus gnavus group. The short-chain fatty acid levels were not significantly different between the groups. The incidence of adverse events was comparable between the groups. DISCUSSION: Oral 1-kestose is well tolerated and provides clinical improvement for patients with mild to moderate ulcerative colitis through modulation of the gut microbiome.

Effect of L-arginine supplement on liver regeneration after partial hepatectomy in rats.

BACKGROUND: Nitric oxide (NO) has been reported to be a key mediator in hepatocyte proliferation during liver regeneration. NO is the oxidative metabolite of L-arginine, and is produced by a family of enzymes, collective termed nitric oxide synthase (NOS). Thus, administration of L-arginine might enhance liver regeneration after a hepatectomy. Another amino acid, L-glutamine, which plays an important role in catabolic states and is a crucial factor in various cellular and organ functions, is widely known to enhance liver regeneration experimentally. Thus, the present study was undertaken to evaluate the effects of an L-arginine supplement on liver regeneration, and to compared this with supplementation with L-glutamine and L-alanine (the latter as a negative control), using a rat partial hepatectomy model. METHODS: Before and after a 70% hepatectomy, rats received one of three amino acid solutions (L-arginine, L-glutamine, or L-alanine). The effects on liver regeneration of the administered solutions were examined by assessment of restituted liver mass, staining for proliferating cell nuclear antigen (PCNA), and total RNA and DNA content 24 and 72 hours after the operation. RESULTS: At 72 hours after the hepatectomy, the restituted liver mass, the PCNA labeling index and the DNA quantity were all significantly higher in the L-arginine and L-glutamine groups than in the control. There were no significant differences in those parameters between the L-arginine and L-glutamine groups, nor were any significant differences found between the L-alanine group and the control. CONCLUSION: Oral supplements of L-arginine and L-glutamine enhanced liver regeneration after hepatectomy in rats, suggesting that an oral arginine supplement can clinically improve recovery after a major liver resection.

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.

Peroxisome proliferator-activated receptor α (PPARα) mRNA expression in human hepatocellular carcinoma tissue and non-cancerous liver tissue.

BACKGROUND: Peroxisome proliferator-activated receptor α (PPARα) regulates lipid metabolism in the liver. It is unclear, however, how this receptor changes in liver cancer tissue. On the other hand, mouse carcinogenicity studies showed that PPARα is necessary for the development of liver cancer induced by peroxisome proliferators, and the relationship between PPARα and the development of liver cancer have been the focus of considerable attention. There have been no reports, however, demonstrating that PPARα is involved in the development of human liver cancer. METHODS: The subjects were 10 patients who underwent hepatectomy for hepatocellular carcinoma. We assessed the expression of PPARα mRNA in human hepatocellular carcinoma tissue and non-cancerous tissue, as well as the expression of target genes of PPARα, carnitine palmitoyltransferase 1A and cyclin D1 mRNAs. We also evaluated glyceraldehyde 3-phosphate dehydrogenase, a key enzyme in the glycolytic system. RESULTS: The amounts of PPARα, carnitine palmitoyltransferase 1A and glyceraldehyde 3-phosphate dehydrogenase mRNA in cancerous sections were significantly increased compared to those in non-cancerous sections. The level of cyclin D1 mRNA tends to be higher in cancerous than non-cancerous sections. Although there was a significant correlation between the levels of PPARα mRNA and cyclin D1 mRNA in both sections, however the correlation was higher in cancerous sections. CONCLUSION: The present investigation indicated increased expression of PPARα mRNA and mRNAs for PPARα target genes in human hepatocellular carcinoma. These results might be associated with its carcinogenesis and characteristic features of energy production.

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.

Bacteroides spp. promotes branched-chain amino acid catabolism in brown fat and inhibits obesity.

The gut microbiome has emerged as a key regulator of obesity; however, its role in brown adipose tissue (BAT) metabolism and association with obesity remain to be elucidated. We found that the levels of circulating branched-chain amino acids (BCAA) and their cognate α-ketoacids (BCKA) were significantly correlated with the body weight in humans and mice and that BCAA catabolic defects in BAT were associated with obesity in diet-induced obesity (DIO) mice. Pharmacological systemic enhancement of BCAA catabolic activity reduced plasma BCAA and BCKA levels and protected against obesity; these effects were reduced in BATectomized mice. DIO mice gavaged with Bacteroides dorei and Bacteroides vulgatus exhibited improved BAT BCAA catabolism and attenuated body weight gain, which were not observed in BATectomized DIO mice. Our data have highlighted a possible link between the gut microbiota and BAT BCAA catabolism and suggest that Bacteroides probiotics could be used for treating obesity.