Neurodegenerative processes accelerated by protein malnutrition and decelerated by essential amino acids in a tauopathy mouse model.

Protein malnutrition is epidemiologically suggested as a potential risk factor for senile dementia, although molecular mechanisms linking dietary proteins and amino acids to neurodegeneration remain unknown. Here, we show that a low-protein diet resulted in down-regulated expression of synaptic components and a modest acceleration of brain atrophy in mice modeling neurodegenerative tauopathies. Notably, these abnormal phenotypes were robustly rescued by the administration of seven selected essential amino acids. The up-regulation of inflammation-associated gene expression and progressive brain atrophy in the tauopathy model were profoundly suppressed by treatment with these essential amino acids without modifications of tau depositions. Moreover, the levels of kynurenine, an initiator of a pathway inducing neuroinflammatory gliosis and neurotoxicity in the brain, were lowered by treatment through inhibition of kynurenine uptake in the brain. Our findings highlight the importance of specific amino acids as systemic mediators of brain homeostasis against neurodegenerative processes.

Protein Deficiency-Induced Behavioral Abnormalities and Neurotransmitter Loss in Aged Mice Are Ameliorated by Essential Amino Acids.

Nutritional epidemiology shows that insufficient protein intake is related to senile dementia. The levels of protein intake in aged people are positively associated with memory function, and elderly people with high protein intake have a low risk of mild cognitive impairment. Although the beneficial roles of protein nutrition in maintaining brain function in aged people are well demonstrated, little is known about the mechanism by which dietary intake of protein affects memory and brain conditions. We fed aged mice a low protein diet (LPD) for 2 months, which caused behavioral abnormalities, and examined the nutritional effect of essential amino acid administration under LPD conditions. The passive avoidance test revealed that LPD mice demonstrated learning and memory impairment. Similarly, the LPD mice showed agitation and hyperactive behavior in the elevated plus maze test. Moreover, LPD mice exhibited decreased concentrations of gamma-aminobutyric acid (GABA), glutamate, glycine, dopamine, norepinephrine, serotonin and aspartate in the brain. Interestingly, oral administration of seven essential amino acids (EAAs; valine, leucine, isoleucine, lysine, phenylalanine, histidine, and tryptophan) to LPD mice, which can be a source of neurotransmitters, reversed those behavioral changes. The oral administration of EAAs restored the brain concentration of glutamate, which is involved in learning and memory ability and may be associated with the observed behavioral changes. Although the details of the link between decreased amino acid and neurotransmitter concentrations and behavioral abnormalities must be examined in future studies, these findings suggest the importance of dietary protein and essential amino acids for maintaining brain function.

Induction of tryptophan hydroxylase in the liver of s.c. tumor model of prostate cancer.

Enhanced degradation of tryptophan (Trp) and thus decreased plasma Trp levels are common in several types of cancers. Although it is well known that Trp catabolism is induced in the tumor microenvironment by the enzymes expressed in cancer cells, immune cells, or both, few studies have examined systemic Trp catabolism in cancer pathophysiology. The present study aimed to evaluate Trp catabolism in both tumor and peripheral tissues using tumor-engrafted Copenhagen rats that were s.c. inoculated with AT-2 rat prostate cancer cells negative for expression of Trp catabolic enzymes. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) metabolomics showed significantly decreased plasma Trp levels in AT-2 engrafted rats, accompanied by increased kynurenine/Trp ratios in spleen and thymus and serotonin levels in liver and thymus. Quantitative PCR and enzymatic activity assays showed indoleamine-2, 3-dioxygenase, an inducible enzyme that catalyzes Trp to kynurenine, was increased in tumor tissues, whereas tryptophan-2,3-dioxygenase, a major Trp catabolic enzyme that regulates systemic level of Trp, tended to be increased in the liver of AT-2 engrafted rats. Furthermore, tryptophan hydroxylase-1 (TPH1), an enzyme that catalyzes the reaction of Trp to serotonin, was significantly increased in liver and spleen of AT-2 engrafted rats. Further histochemical analysis revealed that the induction of TPH1 in the liver could be attributed to infiltration of mast cells. A similar phenomenon was observed with nonneoplastic liver samples from colorectal cancer patients. These results suggested that Trp catabolism toward serotonin synthesis might be induced in peripheral remote tissues in cancer, which could have a pathophysiological effect on cancer.

Overall Similarities and a Possible Factor Affecting Plasma Metabolome Profiles Between Venous and Capillary Blood Samples From 20 Healthy Human Males.

Amino acids and lipids are biomarkers used to assess the presence and severity of disease, as well as the toxicological response to drugs. Although upper-extremity venipuncture is a well-used standard technique, fingertip capillary sampling is a more convenient procedure. Delineating the global differences in amino acid and lipid levels in capillary and venous blood samples is paramount for expanding the application of capillary blood tests in biomarker assays. We recruited 20 healthy male subjects and collected plasma obtained from both fingertip capillary and antecubital venous blood. The samples were analyzed to determine the overall profiles of amino acids and lipids and to test for differences in their levels between both vessel types. The results demonstrated that the differences between capillary and venous blood had a lower impact than interindividual variations; however, trends of separation between them were observed for amino acids. The levels of 5 out of 28 amino acids scored fold changes over 30%, while 9 out of 498 lipids had a fold change over 30%. The time required for fingertip blood collection could be a factor for the differences in 3 metabolites. These findings provide useful information for the application of fingertip capillary blood sampling in biomarker assays.

Facile and Efficient Chemoenzymatic Semisynthesis of Fc-Fusion Compounds for Half-Life Extension of Pharmaceutical Components.

The formation of Fc-fusions, in which biologically active molecules and the Fc fragment of antibodies are linked to each other, is one of the most efficient and successful half-life extension technologies to be developed and applied to peptide and protein pharmaceuticals thus far. Fc-fusion compounds are generally produced by recombinant methods. However, these cannot be applied to artificial middle molecules, such as peptides with non-natural amino acids, unnatural cyclic peptides, or pharmaceutical oligonucleotides. Here, we developed a simple, efficient, semisynthetic method for Fc-fusion production involving our previously developed enzymatic N-terminal extension reaction (i.e., NEXT-A reaction) and strain-promoted azide-alkyne cycloaddition, achieving quantitative conversion and high selectivity for the N-terminus of the Fc protein. An Fc-fusion compound prepared by this method showed comparable biological activity to that of the original peptide and a long-circulating plasma half-life. Thus, the proposed method is potentially applicable for the conjugation of a wide range of pharmaceutical components.

Association between plasma α-aminobutyric acid and depressive symptoms in older community-dwelling adults in Japan.

AIM: To examine the association between depressive symptoms and plasma amino acid related metaboli in older adults. METHODS: A total of 152 older adults aged ≥65 years, residing in Niigata, Japan, were used for analysis. We evaluated depressive symptoms using the Geriatric Depression Scale-15, which has been validated in older community-dwelling individuals, and used a cut off score of ≥5 to classify participants as having depressive symptoms. We used high-performance liquid chromatography-electrospray ionization mass spectrometry to measure the concentrations of plasma amino acid-related metabolites, and carried out logistic regression analysis to assess the association between depressive symptoms and plasma amino acid-related metabolites. RESULTS: Of the 119 older adults (mean age 76.3 years) included in the analysis, 22 were classified as having depressive symptoms (depressive group). There were no significant differences in physical and cognitive impairments between participants in the depressive and non-depressive groups. The plasma α-aminobutyric acid (AABA) level was significantly lower in the depressive group than in the non-depressive group (P < 0.001). Logistic regression analysis showed the best-fit model, which included AABA, leucine, threonine, hydroxyl proline and histidine levels (area under the receiver operating characteristic curve 0.8346; 95% confidence interval 0.7365-0.9326). In particular, the plasma AABA level was strongly associated with depressive symptoms. CONCLUSIONS: Plasma AABA level is significantly associated with depression symptoms in older community-dwelling adults in Japan. Thus, plasma AABA might serve as a potential marker of depression in older adults aged ≥65 years. Geriatr Gerontol Int 2019; 19: 254-258.

Proposed selection strategy of surrogate matrix to quantify endogenous substances by Japan Bioanalysis Forum DG2015-15.

It is important to select an appropriate surrogate matrix for preparing calibration standards and quality control samples while quantitatively assaying for endogenous substances, because a blank matrix that does not contain the endogenous substance cannot be derived from the species from which the target study samples are collected. This is because the assay results might be affected, depending on the characteristics of the analyte in the surrogate matrix. Our discussion group that participated in the Japan Bioanalysis Forum discussed the recommended selection strategies, focusing on large and small molecules in ligand binding assays and LC-MS, respectively. We established an efficient selection strategy for a surrogate matrix, with simple compositions as the first candidates stated in this article.

Interaction of dynamin I with NAP-22, a neuronal protein enriched in the presynaptic region.

Neurons have well-developed membrane microdomains called "rafts" that are recovered as a detergent-resistant low-density membrane microdomain fraction (DRM). NAP-22 is one of the major protein components of neuronal DRM and localizes in the presynaptic region. In order to know the role of NAP-22 in the synaptic transmission, NAP-22 binding proteins in the cytosol were searched with an affinity screening with NAP-22 as a bait and several protein bands were detected. Using mass-analysis and western blotting, one of the main band of ∼90 kDa was identified as dynamin I. The GTPase activity of dynamin I was partly inhibited by NAP-22 expressed in bacteria and this inhibition was recovered by the addition of calmodulin, a NAP-22 binding protein. The GTPase activity of dynamin was known to be activated with acidic membrane lipids such as phosphatidylserine and the addition of NAP-22, a phosphatidylserine binding protein, inhibited the activation of the GTPase by this lipid. Since NAP-22 localizes on the presynaptic plasma membrane and on synaptic vesicles, these results suggest the participation of NAP-22 in the membrane cycling through binding to dynamin and acidic membrane lipids at the presynaptic region.

Inhibitory effect of several sphingolipid metabolites on calcineurin.

Neurons have well-developed membrane microdomains called "rafts" that are recovered as a detergent-resistant membrane microdomain fraction (DRM). NAP-22 is one of the major protein components of neuronal DRM. In a previous study, we showed that DRM-derived NAP-22 binds ganglioside and the inhibitory effect of ganglioside to calcineurin (CaN), a neuron-enriched calmodulin-regulated phosphoprotein phosphatase. Considering the important roles of CaN in neurons, identification of other cellular regulators of CaN could be a good clue to understand the molecular background of neuronal function. In this study, we screened the effect of several membrane lipid-derived molecules on the CaN activity and found sphingosine and some sphingosine-derived metabolites such as sphingosylphosphorylcholine, galactosylsphingosine (psychosine), and glucosylsphingosine, have inhibitory effect on CaN through the interaction with calmodulin.

Slc3a2 Mediates Branched-Chain Amino-Acid-Dependent Maintenance of Regulatory T Cells.

Foxp3+ regulatory T (Treg) cells, which suppress immune responses, are highly proliferative in vivo. However, it remains unclear how the active replication of Treg cells is maintained in vivo. Here, we show that branched-chain amino acids (BCAAs), including isoleucine, are required for maintenance of the proliferative state of Treg cells via the amino acid transporter Slc3a2-dependent metabolic reprogramming. Mice fed BCAA-reduced diets showed decreased numbers of Foxp3+ Treg cells with defective in vivo proliferative capacity. Mice lacking Slc3a2 specifically in Foxp3+ Treg cells showed impaired in vivo replication and decreased numbers of Treg cells. Slc3a2-deficient Treg cells showed impaired isoleucine-induced activation of the mTORC1 pathway and an altered metabolic state. Slc3a2 mutant mice did not show an isoleucine-induced increase of Treg cells in vivo and exhibited multi-organ inflammation. Taken together, these findings demonstrate that BCAA controls Treg cell maintenance via Slc3a2-dependent metabolic regulation.

The role of Kupffer cells in carbon tetrachloride intoxication in mice.

Carbon tetrachloride (CCl(4))-induced acute hepatitis is assumed to involve two phases. The initial phase, initiated within 2 h after CCl(4) administration, involves the generation of reactive oxygen species. The second phase is assumed to start about 8 h subsequent to CCl(4) administration and involves the oxidant-induced activation of Kupffer cells, which release various pro-inflammatory mediators such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6). We investigated the role of Kupffer cells during CCl(4) intoxication using Nucling-knockout mice (the KO group), in which the number of Kupffer cells is largely reduced. Plasma alanine transaminase and aspartate transaminase levels demonstrated that the liver necrosis during the second phase was significantly alleviated in the KO group compared with that in the wild-type mice (the WT group). Plasma TNF-α concentrations in the WT group significantly increased 24 h after CCl(4) intoxication, whereas those in the KO group did not significantly increase. Plasma IL-6 levels also significantly increased in the WT group 24 h after CCl(4) administration, but those in the KO group did not increase at any time point. These results indicated that excess reactions of Kupffer cells, once primed by oxidants, were involved in the exacerbation of oxidative stress and liver damage during the second phase of CCl(4) intoxication.

Studies of the toxicological potential of capsinoids, XII: pharmacokinetic study of capsinoid-containing CH-19 Sweet extract in rats.

Pharmacokinetics of the main capsinoid components of CH-19 Sweet extract (capsiate, dihydrocapsiate, and nordihydrocapsiate) were investigated in rats receiving a single gavage dose of extract containing 10 or 100 mg of capsinoids per kilogram in medium-chain triglyceride. Resultant blood levels of these capsinoids and a capsinoid metabolite, vanillyl alcohol, were measured in portal vein and systemic blood. Capsinoids were never detected. Portal compartment vanillyl alcohol concentrations and area under the plasma concentration versus time curve increased approximately with dose, whereas the time to maximum concentration of vanillyl alcohol was independent of dose (30 minutes post dosing), suggesting that precipitation in the stomach or intestines was unlikely. Vanillyl alcohol levels were just barely detectable in systemic plasma (5 minutes post dosing). Significant levels of vanillyl alcohol conjugates, sulfate, and glucuronide were detected in the systemic blood. Given that the orally administered capsinoids were never detected in the portal vein or systemic circulation, these compounds must be broken down (chemically or enzymatically) to vanillyl alcohol.

Studies of the toxicological potential of capsinoids: X. Safety assessment and pharmacokinetics of capsinoids in healthy male volunteers after a single oral ingestion of CH-19 Sweet extract.

The safety and pharmacokinetics of capsinoids, physiologically active ingredients of CH-19 Sweet extract, were investigated in 16 healthy male volunteers following a single oral ingestion of CH-19 Sweet extract. The study subjects consumed soft gel capsules containing either capsinoids (15 or 30 mg/person) or placebo. Capsinoids were well tolerated, and no clinically significant changes in physical examinations, blood pressure, heart rate, body temperature, electrocardiogram, hematology, blood chemistry, and urinalysis were observed at either the 15 or 30 mg dose. Body temperature tended to increase after the ingestion of capsinoids, but remained within the normal range. Plasma levels of capsinoids and their metabolite, vanillyl alcohol, were below the lower limit of quantitation. In addition, some study subjects showed increases in urinary excretion of 3-methoxy-4-hydroxyphenylglycol that, when compared to the group receiving the placebo, did not achieve statistical significance.