Gamma-aminobutyric acid as a potential postbiotic mediator in the gut-brain axis.

Gamma-aminobutyric acid (GABA) plays a crucial role in the central nervous system as an inhibitory neurotransmitter. Imbalances of this neurotransmitter are associated with neurological diseases, such as Alzheimer's and Parkinson's disease, and psychological disorders, including anxiety, depression, and stress. Since GABA has long been believed to not cross the blood-brain barrier, the effects of circulating GABA on the brain are neglected. However, emerging evidence has demonstrated that changes in both circulating and brain levels of GABA are associated with changes in gut microbiota composition and that changes in GABA levels and microbiota composition play a role in modulating mental health. This recent research has raised the possibility that GABA may be a potent mediator of the gut-brain axis. This review article will cover up-to-date information about GABA-producing microorganisms isolated from human gut and food sources, explanation why those microorganisms produce GABA, food factors inducing gut-GABA production, evidence suggesting GABA as a mediator linking between gut microbiota and mental health, including anxiety, depression, stress, epilepsy, autism spectrum disorder, and attention deficit hyperactivity disorder, and novel information regarding homocarnosine-a predominant brain peptide that is a putative downstream mediator of GABA in regulating brain functions. This review will help us to understand how the gut microbiota and GABA-homocarnosine metabolism play a significant role in brain functions. Nonetheless, it could support further research on the use of GABA production-inducing microorganisms and food factors as agents to treat neurological and psychological disorders.

Relationship of Low Vitamin B6 Status with Sarcopenia, Frailty, and Mortality: A Narrative Review.

Marginal vitamin B6 (B6) deficiency is a widespread global concern. Inadequate B6 levels have been linked to an increased risk of age-related chronic diseases such as cardiovascular diseases and cancers. In recent years, the growing concern over sarcopenia (the age-related loss of muscle mass and strength) and frailty (a decline in physiological resilience and increased vulnerability associated with aging) is particularly relevant due to the emergence of super-aged societies in developed countries. Notably, among the thirty-one studies included in this review, twenty-five showed a significant association of B6 status with sarcopenia, frailty, and all-cause mortality in adults (p < 0.05), while six showed no association. Emerging studies have suggested novel mechanisms underlying this association. These mechanisms involve P2X7 receptor-mediated NLRP3 inflammasome signaling, AMPK signaling, PD-L1 signaling, and satellite cell-mediated myogenesis. Furthermore, the modulation of PLP-dependent enzymes due to B6 deficiency is associated with impaired metabolic processes, affecting energy utilization, imidazole peptide production, and hydrogen sulfide production, as well as the kynurenine pathway, all of which play vital roles in skeletal muscle health and pathophysiology. This narrative review provides an up-to-date assessment of our current understanding of the potential role of nutritional B6 status in combating sarcopenia, frailty, and mortality.

Satellite cell content and muscle regeneration in a mouse model of NAFLD.

OBJECTIVES: Muscle wasting is a common complication in patients with nonalcoholic fatty liver disease (NAFLD). In this study, we investigated the effect of NAFLD on satellite cell (SC) content and skeletal muscle repair. METHODS: Male CD-1 mice fed a choline-deficient diet for 4 wk were used as an NAFLD model. We performed histologic and mRNA expression analyses, immunochemical staining with single muscle fibers to assess the effect of NAFLD on muscle Pax7+ SCs, and muscle regeneration by intramuscular injection of cardiotoxin. RESULTS: We found that the total number of Pax7+ SCs in the extensor digitorum longus and tibialis anterior muscles of mice with NAFLD was significantly decreased when compared with that in the control group, in which the depletion of the SC pool possibly impaired muscle regeneration, as evidenced by the smaller size of the regenerating myofibers. Importantly, we found that NAFLD significantly impaired the differentiation ability of SCs, as shown by a decreased number of SCs expressing a myogenic marker, MyoD. Finally, this study indicated that molecular mechanisms underlying a decline in SC numbers may be attributed to the upregulation of proinflammatory cytokines (tumor necrosis factor α [TNFα]) and an oxidative stress marker (NADPH oxidase-2 [NOX2\) in mice with NAFLD. CONCLUSIONS: The findings demonstrate that a decrease in SC content in the skeletal muscle is an important factor that contributes to muscle wasting in NAFLD. Thus, preservation of the muscle SC pool is a potential therapeutic strategy to reduce NAFLD-associated muscle wasting.

Serum Amyloid A3 Promoter-Driven Luciferase Activity Enables Visualization of Diabetic Kidney Disease.

The early detection of diabetic nephropathy (DN) in mice is necessary for the development of drugs and functional foods. The purpose of this study was to identify genes that are significantly upregulated in the early stage of DN progression and develop a novel model to non-invasively monitor disease progression within living animals using in vivo imaging technology. Streptozotocin (STZ) treatment has been widely used as a DN model; however, it also exhibits direct cytotoxicity to the kidneys. As it is important to distinguish between DN-related and STZ-induced nephropathy, in this study, we compared renal responses induced by the diabetic milieu with two types of STZ models: multiple low-dose STZ injections with a high-fat diet and two moderate-dose STZ injections to induce DN. We found 221 genes whose expression was significantly altered during DN development in both models and identified serum amyloid A3 (Saa3) as a candidate gene. Next, we applied the Saa3 promoter-driven luciferase reporter (Saa3-promoter luc mice) to these two STZ models and performed in vivo bioluminescent imaging to monitor the progression of renal pathology. In this study, to further exclude the possibility that the in vivo bioluminescence signal is related to renal cytotoxicity by STZ treatment, we injected insulin into Saa3-promoter luc mice and showed that insulin treatment could downregulate renal inflammatory responses with a decreased signal intensity of in vivo bioluminescence imaging. These results strongly suggest that Saa3 promoter activity is a potent non-invasive indicator that can be used to monitor DN progression and explore therapeutic agents and functional foods.

Comparative study on molecular mechanism of diabetic myopathy in two different types of streptozotocin-induced diabetic models.

AIMS: Streptozotocin (STZ)-induced diabetic animal models have been widely used to study diabetic myopathy; however, non-specific cytotoxic effects of high-dose STZ have been discussed. The purpose of this study was to compare diabetic myopathy in a high-STZ model with another well-established STZ model with reduced cytotoxicity (high-fat diet (HFD) and low-dose STZ) and to identify mechanistic insights underlying diabetic myopathy in STZ models that can mimic perturbations observed in human patients with diabetic myopathy. MAIN METHODS: Male C57BL6 mice were injected with a single high dose of STZ (180 mg/kg, High-STZ) or were given HFD plus low-dose STZ injection (STZ, 55 mg/kg/day, five consecutive days, HFD/STZ). We characterized diabetic myopathy by histological and immunochemical analyses and conducted gene expression analysis. KEY FINDINGS: The high-STZ model showed a significant reduction in tibialis anterior myofiber size along with decreased satellite cell content and downregulation of inflammation response and collagen gene expression. Interestingly, blood corticosteroid levels were significantly increased in the high-STZ model, which was possibly related to lowered inflammation response-related gene expression. Further analyses using the HFD/STZ model showed downregulation of gene expression related to mitochondrial functions accompanied by a significant decrease in ATP levels in the muscles. SIGNIFICANCE: The high-STZ model is suitable for studies regarding not only severe diabetic myopathy with excessive blood glucose but also negative impact of glucocorticoids on skeletal muscles. In contrast, the HFD/STZ model is characterized by higher immune responses and lower ATP production, which also reflects the pathologies observed in human diabetic patients.

Emerging cardioprotective mechanisms of vitamin B6: a narrative review.

Although overt vitamin B6 deficiency is rare, marginal vitamin B6 deficiency is frequent and occurs in a consistent proportion of the population. The marginal vitamin B6 deficiency appears to relate to an increased risk of inflammation-related diseases, such as cardiovascular diseases and cancers. Of all the cardiovascular diseases, heart failure is a complex clinical syndrome associated with a high mortality rate. So far, information regarding the cardioprotective mechanisms of vitamin B6 has been limited. Meanwhile, recent studies have revealed that vitamin B6 treatment increases cardiac levels of imidazole dipeptides (e.g., carnosine, anserine, and homocarnosine), histamine, and γ-aminobutyric acid (GABA) and suppresses P2X7 receptor-mediated NLRP3 inflammasome. These modulations may imply potential cardioprotective mechanisms of vitamin B6. These modulations may also be involved in the underlying mechanisms through which vitamin B6 suppresses oxidative stress and inflammation. This review provides an up-to-date evaluation of our current understanding of the cardioprotective mechanisms of vitamin B6.

Satellite Cells Exhibit Decreased Numbers and Impaired Functions on Single Myofibers Isolated from Vitamin B6-Deficient Mice.

Emerging research in human studies suggests an association among vitamin B6, sarcopenia, and muscle strength. However, very little is known regarding its potential role at the cellular level, especially in muscle satellite cells. Therefore, to determine whether vitamin B6 affects the satellite cells, we isolated single myofibers from muscles of vitamin B6-deficient and vitamin B6-supplemented mice. Subsequently, we subjected them to single myofiber culture and observed the number and function of the satellite cells, which remained in their niche on the myofibers. Prior to culture, the vitamin B6-deficient myofibers exhibited a significantly lower number of quiescent satellite cells, as compared to that in the vitamin B6-supplemented myofibers, thereby suggesting that vitamin B6 deficiency induces a decline in the quiescent satellite cell pool in mouse muscles. After 48 and 72 h of culture, the number of proliferating satellite cells per cluster was similar between the vitamin B6-deficient and -supplemented myofibers, but their numbers decreased significantly after culturing the myofibers in vitamin B6-free medium. After 72 h of culture, the number of self-renewing satellite cells per cluster was significantly lower in the vitamin B6-deficient myofibers, and the vitamin B6-free medium further decreased this number. In conclusion, vitamin B6 deficiency appears to reduce the number of quiescent satellite cells and suppress the proliferation and self-renewal of satellite cells during myogenesis.

Fermented date residue extract mix containing gamma-aminobutyric acid augments the immune function of mouse splenocytes.

An extract of date (fruit of a palm tree) residue plus food-grade glutamate, acetic acid, and yeast extract (date residue extract mix, DREM) has been successfully fermented with using Lactobacillus brevis JCM 1059T to produce gamma-aminobutyric acid (GABA). Here, mouse splenocytes were found to be viable when supplemented with DREM and fermented DREM containing GABA (fDREM). The addition of DREM and fDREM resulted in the secretion of tumor necrosis factor (TNF)-α from the splenocytes, fDREM being more effective than DREM. The TNF-α secretion with DREM was elevated by exogenous addition of GABA and that with fDREM was in part mediated via A-type GABA receptors. Contrary to general understanding of the suppressive effects of GABA on various biological functions, our findings suggest that GABA-containing fDREM arguments the immune function as a food and pharmaceutical material.

Liver choline metabolism and gene expression in choline-deficient mice offspring differ with gender.

Choline is an important nutrient during pregnancy and lactation. Maternal choline deficiency in CD-1 mice lowers liver betaine levels in male offspring. By contrast, it increases elovl3 and vanin-1 mRNA levels in female offspring. Taken together, these observations suggest gender-specific responses to a choline-deficient diet.

Mungbean seed coat water extract inhibits inflammation in LPS-induced acute liver injury mice and LPS-stimulated RAW 246.7 macrophages via the inhibition of TAK1/IκBα/NF-κB.

Inflammation plays an important role in pathogenesis and progression of many chronic diseases. Although, anti-inflammatory activities of mungbean have been suggested, the underlying mechanism have not been fully understood. The present study aimed to reveal the anti-inflammatory effects of mungbean seed coat water extract (MSWE) in lipopolysaccharide (LPS)-stimulated inflammation in RAW 246.7 macrophages and LPS-induced acute liver injury mice. MSWE pretreatment downregulated the elevated expression of inflammatory markers induced by LPS in the transcriptional and protein level. MSWE inhibited NF-κB activation through the suppression of phosphorylated p65 subunit, IκBα degradation, and transforming growth factor-ß-activated kinases 1 (TAK1) phosphorylation in LPS-stimulated RAW 246.7 cells. Vitexin, the major flavonoid in MSWE showed similar effects. In in vivo experiments, we found that oral administration of MSWE downregulated iNOS expression in LPS-induced acute liver injury mice. The mRNA expression of inflammatory markers and macrophage infiltration was also decreased in the livers. Collectively, MSWE exerts anti-inflammatory role, in part possibly through its active compound vitexin, by inhibiting NF-κB activation via inhibition of TAK1 phosphorylation and IκBα degradation. This suggests that MSWE is beneficial to combat various inflammatory diseases.

Dietary GABA induces endogenous synthesis of a novel imidazole peptide homocarnosine in mouse skeletal muscles.

Carnosine (ß-alanyl-L-histidine) is an imidazole dipeptide present at high concentrations in skeletal muscles, where it plays a beneficial role. However, oral intake of carnosine or ß-alanine to increase skeletal muscle carnosine levels has disadvantages such as low efficiency and side effects. Therefore, we proposed homocarnosine (γ-aminobutyryl-L-histidine) as a novel alternative imidazole peptide for skeletal muscle based on its structural similarity to carnosine. To induce endogenous homocarnosine synthesis in skeletal muscles, mice were fed a basal diet mixed with 0, 0.5, 2, or 5% γ-aminobutyric acid (GABA) for 6 weeks. As expected, in the control group (0% GABA), GABA and homocarnosine were present in trace concentrations. Skeletal muscle homocarnosine levels were significantly increased in the 2% and 5% GABA intake groups (tenfold, P < 0.01 and 53-fold, P < 0.01; respectively) relative to those of the control group, whereas 0.5% GABA intake induced no such effect. GABA intake had no effect on the levels of carnosine, anserine, and ß-alanine. Vigabatrin (inhibitor of GABA transaminase (GABA-T)) administration to mice receiving 2% GABA intake for 2 weeks led to GABA-T inhibition in the liver. Subsequently, a 43-fold increase in circulating GABA levels and a tendency increase in skeletal muscle homocarnosine levels were observed. Therefore, skeletal muscle homocarnosine synthesis can be induced by supplying its substrate GABA in tissues. As GABA availability is tightly regulated by GABA-T via GABA degradation, inhibitors of GABA or ß-alanine degradation could be novel potential interventions for increasing skeletal muscle imidazole dipeptides.

Low-Dose Ethanol Has Impacts on Plasma Levels of Metabolites Relating to Chronic Disease Risk in SAMP8 mice.

The effects of low-dose alcohol on experimental animals are unclear. This study examined plasma metabolites in senescence-accelerated mice 8 (SAMP8) given low-dose ethanol, and compared them with aging progress and skeletal muscle strength. Male SAMP8 mice (10-wk-old) were given drinking water containing 0% (control), 1%, 2%, or 5% (v/v) ethanol for 14 wk. Compared with the control group, only mice who consumed 1% ethanol experienced a lower senescence score at 18 and 23 wk, as well as an increased limb grip strength at 21 wk. Plasma metabolites of control, 1% and 2% ethanol groups were analyzed by capillary electrophoresis-time-of-flight mass spectrometry (CE-TOF/MS). Among the 7 metabolites affected by ethanol, notewhorthy is the positive association of the ethanol levels in drinking water with the levels of α-ketoglutarate (antioxidant and anti-inflammatory metabolite) and hippurate (antioxidant and microbial co-metabolite) (p<0.05). Intriguingly, the levels of 2-hydroxyisobutyrate (the biomarker of energy metabolism and microbial co-metabolite) were higher in the 1% ethanol group (p<0.05), but not in the 2% ethanol group as compared to the control. Furthermore, the levels of some of the metabolites affected were correlated with some variables in the grading score of senescence and muscle strength. This study provides a novel insight into how low-dose ethanol in SAMP8 mice modulates the levels of circulating metabolites relating to chronic disease risk.

Consumption of Low-Dose of Ethanol Suppresses Colon Tumorigenesis in 1,2-Dimethylhydrazine-Treated Rats.

The effect of low-dose of ethanol consumption on the development of colon cancer is unclear. This study aimed to investigate the effects of low-dose ethanol (0.5%, 1%, and 2% [v/v] ethanol in drinking water) for 28 wk on colon tumor incidence in rats injected with 1,2-dimethylhydrazine. Body weight, fluid and food consumption, and the total numbers of colon adenomas (mild-, moderate-, and severe-grade dysplasia) per rat were unaffected by ethanol consumption. However, the numbers of severe-grade dysplasia were significantly reduced by 1% ethanol compared with the control (0% ethanol; -93%) but not by 0.5% and 2% ethanol. Although the numbers of total adenocarcinomas were unaffected, those of total of adenomas and adenocarcinomas together were significantly reduced by 0.5% and 1% ethanol (-39% and -41%, respectively). Intriguingly, real-time PCR assay indicated the abundance of cecal Clostridium leptum (a putative immunosuppressor) was the least in rats received 1% ethanol. Furthermore, 1% ethanol markedly increased colonic mRNA of IL-6, a putative suppressor of regulatory T-cells and cytoprotector. This study provides the first evidence for the potential of 1% ethanol, but not 2% ethanol, to prevent colon tumorigenesis in rats, supporting the J-curve hypothesis of the effect of low-dose alcohol on health. Further, the modulation of C. leptum and expression of IL-6, potentially linking to carcinogenesis, by 1% ethanol may provide an insight into the underlying mechanisms of the anti-colon tumor effect.

The serum amyloid A3 promoter-driven luciferase reporter mice is a valuable tool to image early renal fibrosis development and shows the therapeutic effect of glucosyl-hesperidin treatment.

Tubulointerstitial fibrosis is a progressive process affecting the kidneys, causing renal failure that can be life-threatening. Thus, renal fibrosis has become a serious concern in the ageing population; however, fibrotic development cannot be diagnosed early and assessed noninvasively in both patients and experimental animal models. Here, we found that serum amyloid A3 (Saa3) expression is a potent indicator of early renal fibrosis; we also established in vivo Saa3/C/EBPß-promoter bioluminescence imaging as a sensitive and specific tool for early detection and visualization of tubulointerstitial fibrosis. Saa3 promoter activity is specifically upregulated in parallel with tumor necrosis factor α (TNF-α) and fibrotic marker collagen I in injured kidneys. C/EBPß, upregulated in injured kidneys and expressed in tubular epithelial cells, is essential for the increased Saa3 promoter activity in response to TNF-α, suggesting that C/EBPß plays a crucial role in renal fibrosis development. Our model successfully enabled visualization of the suppressive effects of a citrus flavonoid derivative, glucosyl-hesperidin, on inflammation and fibrosis in kidney disease, indicating that this model could be widely used in exploring therapeutic agents for fibrotic diseases.

Feeding Aspergillus protease preparation combined with adequate protein diet to rats increases levels of cecum gut-protective amino acids, partially linked to Bifidobacterium and Lactobacillus.

Our recent study indicated that dietary Aspergillus oryzae-derived protease preparation (AP), through its enzymatic activity, exerted a bifidogenic effect in rats. We hypothesized that dietary AP links to protein degradation and subsequently elevates gut-protective amino acids (AAs) in rats fed adequate protein diet. In this study, dietary AP markedly increased the relative abundance of Bifidobacterium and Lactobacillus and the levels of free threonine, alanine, proline, taurine, ornithine, phenylalanine, cystine, and γ-aminobutyric acid in the cecum contents of rats fed with an adequate protein diet, but not in those fed with a low-protein diet. The elevated AAs, except ornithine and phenylalanine, potentially have gut-related health benefits. Some of the AP-modulated free AAs appeared to be associated with the relative abundance of Bifidobacterium and Lactobacillus. Thus, AP combined with adequate protein diet is likely to increase the levels of cecum beneficial free AAs, which is partially associated with the relative abundance of the probiotics.

Novel metabolic disturbances in marginal vitamin B6-deficient rat heart.

Vitamin B6 deficiency is associated with cardiovascular disease (CVD). Although plasma biomarkers have been proposed, no studies have yet directly profiled heart tissue, and the mechanisms have to be fully defined. Thus, in order to provide better insight into vitamin B6-deficient effects on cardiac functions, we sought to identify the metabolic profile in heart tissue consequent to change in dietary vitamin B6 levels by applying metabolomics. Heart tissues of rats fed a basal diet containing a marginal vitamin B6-deficient, vitamin B6-recommended or vitamin B6-supplemented level were analyzed by metabolomics analysis. Among over 500 detected metabolites, imidazole metabolites including carnosine, anserine, homocarnosine and histamine exhibited the highest decrease upon vitamin B6 deficiency (>-45%, P<.01), along with their precursors ß-alanine, γ-aminobutyric acid (GABA) and 1-methylhistidine. Ornithine was the only metabolite exhibiting an increased level in the vitamin B6-deficient group. Vitamin B6 deficiency significantly attenuated the activity of heart tissue glutamate decarboxylase (GAD), although there was undetectable activity of aspartate decarboxylase (ADC), suggesting that the involvement of vitamin B6 in imidazole metabolite synthesis occurs partly through GABA production by regulating GAD rather than through a straightforward ß-alanine production pathway via ADC in the heart. Notably, vitamin B6 deficiency significantly attenuated citric acid cycle metabolite levels, suggesting cardiac energy metabolism impairment. This study provides a new link between vitamin B6 and cardiac functions, in which marginal vitamin B6 deficiency impairs imidazole and energy metabolism in heart. This newly revealed cardiac metabolic profile may reveal novel molecular targets or foodstuffs for CVD prevention.

GDE5 inhibition accumulates intracellular glycerophosphocholine and suppresses adipogenesis at a mitotic clonal expansion stage.

Mammalian glycerophosphodiesterases (GDEs) were recently shown to be involved in multiple cellular signaling pathways. This study showed that decreased GDE5 expression results in accumulation of intracellular glycerophosphocholine (GPC), showing that GDE5 is actively involved in GPC/choline metabolism in 3T3-L1 adipocytes. Using 3T3-L1 adipocytes, we further studied the biological significance of GPC/choline metabolism during adipocyte differentiation. Inhibition of GDE5 suppressed the formation of lipid droplets, which is accompanied by the decreased expression of adipocyte differentiation markers. We further showed that the decreased GDE5 expression suppressed mitotic clonal expansion (MCE) of preadipocytes. Decreased expression of CTP: phosphocholine cytidylyltransferase (CCTß), a rate-limiting enzyme for phosphatidylcholine (PC) synthesis, is similarly able to inhibit MCE and PC synthesis; however, the decreased GDE5 expression resulted in accumulation of intracellular GPC but did not affect PC synthesis. Furthermore, we showed that mRNAs of proteoglycans and transporters for organic osmolytes are significantly upregulated and that intracellular amino acids and urea levels are altered in response to GDE5 inhibition. Finally, we showed that reduction of GDE5 expression increased lactate dehydrogenase release from preadipocytes. These observations indicate that decreased GDE5 expression can suppress adipocyte differentiation not through the PC pathway but possibly by intracellular GPC accumulation. These results provide insight into the roles of mammalian GDEs and their dependence upon osmotic regulation by altering intracellular GPC levels.

Increased intestinal ethanol following consumption of fructooligosaccharides in rats.

Previous studies have suggested that ethanol is a fermentation product of microflora. However, it is unknown whether this ethanol production is elevated by intake of prebiotics. Prebiotics are considered to enhance the production of short-chain fatty acids (SCFAs) as a fermentation product of beneficial bacteria. In the present study, the effect of fructooligosaccharides (FOS) consumption on intestinal ethanol levels was investigated. Rats were fed a diet with or without 10% FOS for two weeks. Consequently, FOS intake significantly increased ethanol levels per gram of ileum and cecum digesta of the rats (3.5-fold and 1.9-fold, respectively, P<0.01). The numbers of cecum Bifidobacterium (producer of ethanol and lactate) were significantly increased by FOS intake (P<0.05) and correlated with the cecum ethanol levels per gram of cecum (r=0.626, P<0.05). FOS intake also led to a significant increase in the cecum levels of SCFAs, namely lactate, propionate and n-butyrate (P<0.05). Furthermore, ethanol levels were significantly correlated with lactate levels (r=0.691, P<0.01), but not with propionate or n-butyrate levels (r=0.449 and 0.493, respectively, P>0.05). The current study, to the best of our knowledge, is the first to indicate that FOS intake significantly increases the level of intestinal ethanol. Therefore, dietary FOS may affect the intestinal health status of animals by elevating their ethanol levels, without direct ethanol consumption.