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.

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.

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.

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.

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.

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.

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.

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.

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.

1-Kestose supplementation mitigates the progressive deterioration of glucose metabolism in type 2 diabetes OLETF rats.

The fructooligosaccharide 1-kestose cannot be hydrolyzed by gastrointestinal enzymes, and is instead fermented by the gut microbiota. Previous studies suggest that 1-kestose promotes increases in butyrate concentrations in vitro and in the ceca of rats. Low levels of butyrate-producing microbiota are frequently observed in the gut of patients and experimental animals with type 2 diabetes (T2D). However, little is known about the role of 1-kestose in increasing the butyrate-producing microbiota and improving the metabolic conditions in type 2 diabetic animals. Here, we demonstrate that supplementation with 1-kestose suppressed the development of diabetes in Otsuka Long-Evans Tokushima Fatty (OLETF) rats, possibly through improved glucose tolerance. We showed that the cecal contents of rats fed 1-kestose were high in butyrate and harbored a higher proportion of the butyrate-producing genus Anaerostipes compared to rats fed a control diet. These findings illustrate how 1-kestose modifications to the gut microbiota impact glucose metabolism of T2D, and provide a potential preventative strategy to control glucose metabolism associated with dysregulated insulin secretion.

BDK Deficiency in Cerebral Cortex Neurons Causes Neurological Abnormalities and Affects Endurance Capacity.

Branched-chain amino acid (BCAA) catabolism is regulated by its rate-limiting enzyme, branched-chain α-keto acid dehydrogenase (BCKDH), which is negatively regulated by BCKDH kinase (BDK). Loss of BDK function in mice and humans leads to dysregulated BCAA catabolism accompanied by neurological symptoms such as autism; however, which tissues or cell types are responsible for the phenotype has not been determined. Since BDK is highly expressed in neurons compared to astrocytes, we hypothesized that neurons are the cell type responsible for determining the neurological features of BDK deficiency. To test this hypothesis, we generated mice in which BDK deletion is restricted to neurons of the cerebral cortex (BDKEmx1-KO mice). Although BDKEmx1-KO mice were born and grew up normally, they showed clasped hind limbs when held by the tail and lower brain BCAA concentrations compared to control mice. Furthermore, these mice showed a marked increase in endurance capacity after training compared to control mice. We conclude that BDK in neurons of the cerebral cortex is essential for maintaining normal neurological functions in mice, and that accelerated BCAA catabolism in that region may enhance performance in running endurance following training.

Reply to Comment on Watanabe, A.; Kadota, Y.; Yokoyama, H.; Tsuruda, S.; Kamio, R.; Tochio, T.; Shimomura, Y.; Kitaura, Y. Experimental Determination of the Threshold Dose for Bifidogenic Activity of Dietary 1-Kestose in Rats. Foods 2020, 9, 4.

The manuscript entitled "Comment on Experimental Determination of the Threshold Dose for Bifidogenic Activity of Dietary 1-Kestose in Rats" by Shen et al [...].

Branched-chain amino acid supplementation ameliorates angiotensin II-induced skeletal muscle atrophy.

AIMS: Sarcopenia is characterized by muscle mass and strength loss and reduced physical activity. Branched-chain amino acids (BCAAs) were recently described as an activator of protein synthesis via mammalian target of rapamycin (mTOR) signaling for muscle atrophy. In cardiovascular diseases, excessive activation of the renin-angiotensin system may induce an imbalance of protein synthesis and degradation, and this plays a crucial role in muscle atrophy. We investigated the effects of BCAAs on angiotensin II (Ang II)-induced muscle atrophy in mice. MATERIALS AND METHODS: We administered Ang II (1000 ng/kg/min) or vehicle to 10-12-week-old male C57BL/6J mice via subcutaneous osmotic minipumps for 4 weeks with or without BCAA supplementation (3% BCAA in tap water). KEY FINDINGS: The skeletal muscle weight/tibial length and cross-sectional area were smaller in the Ang II mice than the vehicle mice; these changes were induced by an imbalance of protein synthesis and degradation signaling such as Akt/mTOR and MuRF-1/Atrogin-1. Compared to the Ang II mice, the mTOR signaling was significantly activated and Ang II-induced muscle atrophy was ameliorated in the Ang II + BCAA mice, and this attenuated the reduction of exercise capacity. Notably, the decrease of muscle weight/tibial length in the fast-twitch dominant muscles (e.g., the extensor digitorum longus) was significantly ameliorated compared to that in the slow-twitch dominant muscles (e.g., soleus). Histologically, the effect of BCAA was larger in fast-twitch than slow-twitch fibers, which may be related to the difference in BCAA catabolism. SIGNIFICANCE: BCAA supplementation could contribute to the prevention of skeletal muscle atrophy induced by Ang II.

Experimental Determination of the Threshold Dose for Bifidogenic Activity of Dietary 1-Kestose in Rats.

1-Kestose is a non-digestible oligosaccharide consisting of glucose linked to two fructose units. While 1-kestose is not digested in the small intestine of mammals, it is fermented in the ceca and colon, where the growth of bifidobacteria is promoted. In the present study, we assessed the threshold dose of dietary 1-kestose that increased cecal bifidobacterial levels in rats. Rats were fed experimental diets containing 0% to 0.3% 1-kestose for four weeks. The levels of the genus Bifidobacterium and total gut bacteria were significantly increased in cecal samples of rats fed the 0.3% 1-kestose diet. Further, a significant correlation between the dose of 1-kestose and the levels of cecal Bifidobacterium and total gut bacteria was observed. The minimum dose of dietary 1-kestose to induce significant bifidogenic activity in rats was 0.3% by weight in the diet.

Feeding of 1-Kestose Induces Glutathione-S-Transferase Expression in Mouse Liver.

Functional food ingredients, including prebiotics, have been increasingly developed for human health. The improvement of the human intestinal environment is one of their main targets. Fructooligosaccarides (FOS) are oligosaccharide fructans that are well studied and commercialized prebiotics. 1-Kestose, one of the components of FOS, is considered to be a key prebiotic component in FOS. However, to our knowledge, no studies have been reported on the physiological efficacy of 1-Kestose regarding its anti-oxidative activity. In the present study, we examined the effects of dietary 1-Kestose on gene expression of antioxidative enzymes in the liver, kidney and epididymal adipose tissue of mice by quantitative RT-PCR (qRT-PCR). We demonstrated that a 1-Kestose-rich diet increased mRNA and enzymatic activity levels of glutathione-S-transferase (GST) in mouse liver. These results suggest the possibility that dietary 1-Kestose as a prebiotic may enhance antioxidative activity in mice.

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.

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.

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.