Markers for obese and non-obese Type 2 diabetes identified using whole blood metabolomics.

Definitive differences in blood metabolite profiles between obese and non-obese Type 2 diabetes (T2D) have not been established. We performed an LC-MS-based non-targeted metabolomic analysis of whole blood samples collected from subjects classified into 4 types, based on the presence or absence of obesity and T2D. Of the 125 compounds identified, 20, comprising mainly nucleobases and glucose metabolites, showed significant increases or decreases in the T2D group. These included cytidine, UDP-glucuronate, UMP, 6-phosphogluconate, and pentose-phosphate. Among those 20 compounds, 11 enriched in red blood cells (RBCs) have rarely been studied in the context of diabetes, indicating that RBC metabolism is more extensively disrupted than previously known. Correlation analysis revealed that these T2D markers include 15 HbA1c-associated and 5 irrelevant compounds that may reflect diabetic conditions by a different mechanism than that of HbA1c. In the obese group, enhanced protein and fatty acid catabolism causes increases in 13 compounds, including methylated or acetylated amino acids and short-chain carnitines. Our study, which may be considered a pilot investigation, suggests that changes in blood metabolism due to obesity and diabetes are large, but essentially independent.

Decline of ergothioneine in frailty and cognition impairment.

Ergothioneine is a well-known antioxidant that is abundant in both human red blood cells and in fission yeast responding to nutritional stress. In frail elderly people, whose ageing organs undergo functional decline, there is a correlation between ergothioneine levels and cognitive, but not skeletal muscle decline. In patients suffering from dementia, including Alzheimer's disease with hippocampal atrophy, deteriorating cognitive ability is correlated with declining ergothioneine levels. S-methyl-ergothioneine, trimethyl-histidine and three other trimethyl-ammonium compounds also decrease sharply in dementia, whereas compounds such as indoxyl-sulfate and quinolinic acid increase, possibly exacerbating the disease. Using these opposing dementia markers, not only diagnosis, but also therapeutic interventions to mitigate cognitive decline may now become possible.

Reduced uremic metabolites are prominent feature of sarcopenia, distinct from antioxidative markers for frailty.

Due to global aging, frailty and sarcopenia are increasing. Sarcopenia is defined as loss of volume and strength of skeletal muscle in elderlies, while frailty involves multiple domains of aging-related dysfunction, impaired cognition, hypomobility, and decreased social activity. However, little is known about the metabolic basis of sarcopenia, either shared with or discrete from frailty. Here we analyzed comprehensive metabolomic data of human blood in relation to sarcopenia, previously collected from 19 elderly participants in our frailty study. Among 131 metabolites, we identified 22 sarcopenia markers, distinct from 15 frailty markers, mainly including antioxidants, although sarcopenia overlaps clinically with physical frailty. Notably, 21 metabolites that decline in sarcopenia or low SMI are uremic compounds that increase in kidney dysfunction. These comprise TCA cycle, urea cycle, nitrogen, and methylated metabolites. Sarcopenia markers imply a close link between muscle and kidney function, while frailty markers define a state vulnerable to oxidative stress.

Whole-blood metabolomics of dementia patients reveal classes of disease-linked metabolites.

Dementia is caused by factors that damage neurons. We quantified small molecular markers in whole blood of dementia patients, using nontargeted liquid chromatography-mass spectroscopy (LC-MS). Thirty-three metabolites, classified into five groups (A to E), differed significantly in dementia patients, compared with healthy elderly subjects. Seven A metabolites present in plasma, including quinolinic acid, kynurenine, and indoxyl-sulfate, increased. Possibly they act as neurotoxins in the central nervous system (CNS). The remaining 26 compounds (B to E) decreased, possibly causing a loss of support or protection of the brain in dementia. Six B metabolites, normally enriched in red blood cells (RBCs), all contain trimethylated ammonium moieties. These metabolites include ergothioneine and structurally related compounds that have scarcely been investigated as dementia markers, validating the examination of RBC metabolites. Ergothioneine, a potent antioxidant, is significantly decreased in various cognition-related disorders, such as mild cognitive impairment and frailty. C compounds also include some oxidoreductants and are normally abundant in RBCs (NADP+, glutathione, adenosine triphosphate, pantothenate, S-adenosyl-methionine, and gluconate). Their decreased levels in dementia patients may also contribute to depressed brain function. Twelve D metabolites contains plasma compounds, such as amino acids, glycerophosphocholine, dodecanoyl-carnitine, and 2-hydroxybutyrate, which normally protect the brain, but their diminution in dementia may reduce that protection. Seven D compounds have been identified previously as dementia markers. B to E compounds may be critical to maintain the CNS by acting directly or indirectly. How RBC metabolites act in the CNS and why they diminish significantly in dementia remain to be determined.

Human age-declined saliva metabolic markers determined by LC-MS.

Metabolites in human biofluids reflect individual physiological states influenced by various factors. Using liquid chromatography-mass spectrometry (LC-MS), we conducted non-targeted, non-invasive metabolomics using saliva of 27 healthy volunteers in Okinawa, comprising 13 young (30 ± 3 year) and 14 elderly (76 ± 4 year) subjects. Few studies have comprehensively identified age-dependent changes in salivary metabolites. Among 99 salivary metabolites, 21 were statistically age-related. All of the latter decline in abundance with advancing age, except ATP, which increased 1.96-fold in the elderly, possibly due to reduced ATP consumption. Fourteen age-linked and highly correlated compounds function in a metabolic network involving the pentose-phosphate pathway, glycolysis/gluconeogenesis, amino acids, and purines/pyrimidines nucleobases. The remaining seven less strongly correlated metabolites, include ATP, anti-oxidation-related glutathione disulfide, muscle-related acetyl-carnosine, N-methyl-histidine, creatinine, RNA-related dimethyl-xanthine and N-methyl-adenosine. In addition, glutamate and N-methyl-histidine are related to taste, so their decline suggests that the elderly lose some ability to taste. Reduced redox metabolism and muscle activity are suggested by changes in glutathione and acetyl-carnosine. These age-linked salivary metabolites together illuminate a metabolic network that reflects a decline of oral functions during human aging.

Aging markers in human urine: A comprehensive, non-targeted LC-MS study.

Metabolites in human biofluids document the physiological status of individuals. We conducted comprehensive, non-targeted, non-invasive metabolomic analysis of urine from 27 healthy human subjects, comprising 13 young adults (30 ± 3 years) and 14 seniors (76 ± 4 years). Quantitative analysis of 99 metabolites revealed 55 that displayed significant differences in abundance between the two groups. Forty-four did not show a statistically significant relationship with age. These include 13 standard amino acids, 5 methylated, 4 acetylated, and 9 other amino acids, 6 nucleosides, nucleobases, and derivatives, 4 sugar derivatives, 5 sugar phosphates, 4 carnitines, 2 hydroxybutyrates, 1 choline, and 1 ethanolamine derivative, and glutathione disulfide. Abundances of 53 compounds decreased, while 2 (glutathione disulfide, myo-inositol) increased in elderly people. The great majority of age-linked markers were highly correlated with creatinine. In contrast, 44 other urinary metabolites, including urate, carnitine, hippurate, and betaine, were not age-linked, neither declining nor increasing in elderly subjects. As metabolite profiles of urine and blood are quite different, age-related information in urine offers additional valuable insights into aging mechanisms of endocrine system. Correlation analysis of urinary metabolites revealed distinctly inter-related groups of compounds.

Metabolomics of human fasting: new insights about old questions.

Since ancient days, human fasting has been performed for religious or political reasons. More recently, fasting has been employed as an effective therapy for weight reduction by obese people, and numerous studies have investigated the physiology of fasting by obese subjects. Well-established fasting markers (butyrates, BCAAs and carnitines) were considered essential energy substitutes after glycogen storage depletion. However, a recently developed metabolomic approach has unravelled previously unappreciated aspects of fasting. Surprisingly, one-third (44) of 120 metabolites investigated increase during 58 h of fasting, including antioxidative metabolites (carnosine, ophthalmic acid, ergothioneine and urates) and metabolites of entire pathways, such as the pentose phosphate pathway. Signalling metabolites (3-hydroxybutyrate and 2-oxoglutarate) and purines/pyrimidines may also serve as transcriptional modulators. Thus, prolonged fasting activates both global catabolism and anabolism, reprogramming metabolic homeostasis.

Frailty markers comprise blood metabolites involved in antioxidation, cognition, and mobility.

As human society ages globally, age-related disorders are becoming increasingly common. Due to decreasing physiological reserves and increasing organ system dysfunction associated with age, frailty affects many elderly people, compromising their ability to cope with acute stressors. Frail elderly people commonly manifest complex clinical symptoms, including cognitive dysfunction, hypomobility, and impaired daily activity, the metabolic basis of which remains poorly understood. We applied untargeted, comprehensive LC-MS metabolomic analysis to human blood from 19 frail and nonfrail elderly patients who were clinically evaluated using the Edmonton Frail Scale, the MoCA-J for cognition, and the TUG for mobility. Among 131 metabolites assayed, we identified 22 markers for frailty, cognition, and hypomobility, most of which were abundant in blood. Frailty markers included 5 of 6 markers specifically related to cognition and 6 of 12 markers associated with hypomobility. These overlapping sets of markers included metabolites related to antioxidation, muscle or nitrogen metabolism, and amino acids, most of which are decreased in frail elderly people. Five frailty-related metabolites that decreased-1,5-anhydroglucitol, acetyl-carnosine, ophthalmic acid, leucine, and isoleucine-have been previously reported as markers of aging, providing a metabolic link between human aging and frailty. Our findings clearly indicate that metabolite profiles efficiently distinguish frailty from nonfrailty. Importantly, the antioxidant ergothioneine, which decreases in frailty, is neuroprotective. Oxidative stress resulting from diminished antioxidant levels could be a key vulnerability for the pathogenesis of frailty, exacerbating illnesses related to human aging.

Diverse metabolic reactions activated during 58-hr fasting are revealed by non-targeted metabolomic analysis of human blood.

During human fasting, metabolic markers, including butyrates, carnitines, and branched-chain amino acids, are upregulated for energy substitution through gluconeogenesis and use of stored lipids. We performed non-targeted, accurate semiquantitative metabolomic analysis of human whole blood, plasma, and red blood cells during 34-58 hr fasting of four volunteers. During this period, 44 of ~130 metabolites increased 1.5~60-fold. Consistently fourteen were previously reported. However, we identified another 30 elevated metabolites, implicating hitherto unrecognized metabolic mechanisms induced by fasting. Metabolites in pentose phosphate pathway are abundant, probably due to demand for antioxidants, NADPH, gluconeogenesis and anabolic metabolism. Global increases of TCA cycle-related compounds reflect enhanced mitochondrial activity in tissues during fasting. Enhanced purine/pyrimidine metabolites support RNA/protein synthesis and transcriptional reprogramming, which is promoted also by some fasting-related metabolites, possibly via epigenetic modulations. Thus diverse, pronounced metabolite increases result from greatly activated catabolism and anabolism stimulated by fasting. Anti-oxidation may be a principal response to fasting.

S-Adenosylmethionine Synthetase Is Required for Cell Growth, Maintenance of G0 Phase, and Termination of Quiescence in Fission Yeast.

S-adenosylmethionine is an important compound, because it serves as the methyl donor in most methyl transfer reactions, including methylation of proteins, nucleic acids, and lipids. However, cellular defects in the genetic disruption of S-adenosylmethionine synthesis are not well understood. Here, we report the isolation and characterization of temperature-sensitive mutants of fission yeast S-adenosylmethionine synthetase (Sam1). Levels of S-adenosylmethionine and methylated histone H3 were greatly diminished in sam1 mutants. sam1 mutants stopped proliferating in vegetative culture and arrested specifically in G2 phase without cell elongation. Furthermore, sam1 mutants lost viability during nitrogen starvation-induced G0 phase quiescence. After release from the G0 state, sam1 mutants could neither increase in cell size nor re-initiate DNA replication in the rich medium. Sam1 is thus required for cell growth and proliferation, and maintenance of and exit from quiescence. sam1 mutants lead to broad cellular and drug response defects, as expected, since S. pombe contains more than 90 S-adenosylmethionine-dependent methyltransferases.

The putative ceramide-conjugation protein Cwh43 regulates G0 quiescence, nutrient metabolism and lipid homeostasis in fission yeast.

Cellular nutrient states control whether cells proliferate, or whether they enter or exit quiescence. Here, we report characterizations of fission yeast temperature-sensitive (ts) mutants of the evolutionarily conserved transmembrane protein Cwh43, and explore its relevance to utilization of glucose, nitrogen source and lipids. GFP-tagged Cwh43 localizes at ER associated with the nuclear envelope and the plasma membrane, as in budding yeast. We found that cwh43 mutants failed to divide in low glucose and lost viability during quiescence under nitrogen starvation. In cwh43 mutants, comprehensive metabolome analysis demonstrated dramatic changes in marker metabolites that altered under low glucose and/or nitrogen starvation, although cwh43 cells apparently consumed glucose in the culture medium. Furthermore, we found that cwh43 mutant cells had elevated levels of triacylglycerols (TGs) and coenzyme A, and that they accumulated lipid droplets. Notably, TG biosynthesis was required to maintain cell division in the cwh43 mutant. Thus, Cwh43 affects utilization of glucose and nitrogen sources, as well as storage lipid metabolism. These results may fit a notion developed in budding yeast stating that Cwh43 conjugates ceramide to glycosylphosphatidylinositol (GPI)-anchored proteins and maintains integrity of membrane organization.

Genetic defects in SAPK signalling, chromatin regulation, vesicle transport and CoA-related lipid metabolism are rescued by rapamycin in fission yeast.

Rapamycin inhibits TOR (target of rapamycin) kinase, and is being used clinically to treat various diseases ranging from cancers to fibrodysplasia ossificans progressiva. To understand rapamycin mechanisms of action more comprehensively, 1014 temperature-sensitive (ts) fission yeast (Schizosaccharomyces pombe) mutants were screened in order to isolate strains in which the ts phenotype was rescued by rapamycin. Rapamycin-rescued 45 strains, among which 12 genes responsible for temperature sensitivity were identified. These genes are involved in stress-activated protein kinase (SAPK) signalling, chromatin regulation, vesicle transport, and CoA- and mevalonate-related lipid metabolism. Subsequent metabolome analyses revealed that rapamycin upregulated stress-responsive metabolites, while it downregulated purine biosynthesis intermediates and nucleotide derivatives. Rapamycin alleviated abnormalities in cell growth and cell division caused by sty1 mutants (Δsty1) of SAPK. Notably, in Δsty1, rapamycin reduced greater than 75% of overproduced metabolites (greater than 2× WT), like purine biosynthesis intermediates and nucleotide derivatives, to WT levels. This suggests that these compounds may be the points at which the SAPK/TOR balance regulates continuous cell proliferation. Rapamycin might be therapeutically useful for specific defects of these gene functions.

Design and synthesis of curcumin derivatives as tau and amyloid ß dual aggregation inhibitors.

Alzheimer's disease (AD) is the most common form of dementia. In an AD patient's brain, senile plaques and neurofibrillary tangles, the abnormal aggregates of amyloid ß (Aß) peptide and tau protein, are observed as the two major hallmarks of this disease. To develop a new drug for treatment of AD, we have designed and synthesized a series of curcumin derivatives and evaluated their inhibitory activities against both tau and Aß aggregation. In this study, we describe the development of the more potent aggregation inhibitor 3-[(1E)-2-(1H-indol-6-yl)ethenyl]-5-[(1E)-2-[2-methoxy-4-(2-pyridylmethoxy) phenyl] ethenyl]-1H-pyrazole (compound 4, PE859). This compound has a better pharmacokinetic profile and pharmacological efficacy in vivo than curcumin, making it suitable as a drug for AD.

Cleavable linker for photo-cross-linked small-molecule affinity matrix.

The introduction of a cleavable site in a photoactivatable linker, which is used to immobilize small molecules on an affinity matrix via a site-nonselective carbene addition/insertion reaction, makes it possible to verify the presence of the immobilized small molecule on the affinity matrix. It also permits the efficient detection of proteins covalently bound to the immobilized small molecule.

Deamino-hydroxy-phoslactomycin B, a biosynthetic precursor of phoslactomycin, induces myeloid differentiation in HL-60 cells.

During the screening for novel differentiation inducers, we found that a culture broth of Streptomyces sp. HK-803 induced myeloid differentiation of HL-60 cells. The active substance was identified as deamino-hydroxy-phoslactomycin B (HPLM) by mass spectrometry, and synthesized HPLM also induced the differentiation of HL-60 cells. HPLM showed greater inhibition of protein phosphatase 2A (PP2A) activity than phoslactomycin B (PLMB); however, PLMB and okadaic acid did not induce differentiation. Moreover, treatment with ATRA and 1alpha, 25(OH)2D3 induced retinoic acid receptor-beta and 1alpha, 25(OH)2D3 24-hydroxylase, respectively, whereas HPLM did not, suggesting that HPLM is a novel differentiation inducer.

Phoslactomycin targets cysteine-269 of the protein phosphatase 2A catalytic subunit in cells.

According to the chemical genetic approach, small molecules that bind directly to proteins are used to analyze protein function, thereby enabling the elucidation of complex mechanisms in mammal cells. Thus, it is very important to identify the molecular targets of compounds that induce a unique phenotype in a target cell. Phoslactomycin A (PLMA) is known to be a potent inhibitor of protein Ser/Thr phosphatase 2A (PP2A); however, the inhibitory mechanism of PP2A by PLMA has not yet been elucidated. Here, we demonstrated that PLMA directly binds to the PP2A catalytic subunit (PP2Ac) in cells by using biotinylated PLMA, and the PLMA-binding site was identified as the Cys-269 residue of PP2Ac. Moreover, we revealed that the Cys-269 contributes to the potent inhibition of PP2Ac activity by PLMA. These results suggest that PLMA is a PP2A-selective inhibitor and is therefore expected to be useful for future investigation of PP2A function in cells.

Structure-based design of a selective heparanase inhibitor as an antimetastatic agent.

Heparanase is an endo-beta-D-glucuronidase that degrades heparan sulfate glycosaminoglycans in the extracellular matrix and the basement membrane and is well known to be involved in tumor cell invasion and angiogenesis. We have focused on heparanase as a target for antitumor agents, especially antimetastatic agents. (R)-3-hexadecanoyl-5-hydroxymethyltetronic acid (RK-682) was found to display an inhibitory activity against heparanase in our screening of natural sources. Because RK-682 has been reported to show inhibitory activities against several enzymes, we have tried to develop selective heparanase inhibitors using the method of rational drug design. Based on the structure of the heparanase/RK-682 complex, we speculated that selective inhibitory activity against heparanase could be acquired by arylalkylation, namely, by benzylation of the 4-position of RK-682. Among the rationally designed 4-alkyl-RK-682 derivatives, 4-benzyl-RK-682 has been found to possess a selective inhibitory activity for heparanase (IC50 for heparanase, 17 micromol/L; IC50 for other enzymes, >100 micromol/L). 4-Benzyl-RK-682 also inhibited the invasion and migration of human fibrosarcoma HT1080 cells (IC50 for invasion, 1.5 micromol/L; IC50 for migration, 3.0 micromol/L). On the other hand, RK-682 had no inhibitory effect on the invasion and migration of HT1080 cells at doses of up to 100 micromol/L.