Effects of compound-326, a selective delta-5 desaturase inhibitor, in ApoE knockout mice with two different protocols for atherosclerosis development.

PURPOSE: We previously confirmed its anti-atherosclerotic effects by pre-treatment with compound-326, a selective delta-5 desaturase (D5D) inhibitor, in Western diet-fed ApoE knockout mice. In the present study, we evaluated effects of compound-326 in ApoE knockout mice with two different protocols for atherosclerosis development. METHODS: In a post-treatment protocol, where the compound treatment started after 10 weeks pre-feeding of Western diet, compound-326 (1 and 3 mg/kg/day, p.o. for 12 weeks) significantly reduced the atherosclerotic lesion area in the aorta (24% reduction at 3 mg/kg/day). In another protocol using Paigen diet (containing 12.5% cholesterol and 5% sodium cholate), compound-326 (3 and 10 mg/kg/day, p.o. for 7 weeks) also significantly reduced the lesion area (36% reduction at 3 mg/kg/day). RESULTS: In both protocols, Compound-326 significantly reduced the hepatic ratio of arachidonic acid to dihomo-γ-linolenic acid, blood inflammatory eicosanoid production and plasma soluble intercellular adhesion molecule 1 (sICAM-1) levels, similarly to the previous pre-treatment study. CONCLUSIONS: Compound-326 exerted anti-atherosclerotic effects in ApoE knockout mice with the two different protocols for atherosclerosis development further supporting D5D inhibition as a promising strategy in treating atherosclerosis.

Intersubject and Intrasubject Variability of Potential Plasma and Urine Metabolite and Protein Biomarkers in Healthy Human Volunteers.

A limited understanding of intersubject and intrasubject variability hampers effective biomarker translation from in vitro/in vivo studies to clinical trials and clinical decision support. Specifically, variability of biomolecule concentration can play an important role in interpretation, power analysis, and sampling time designation. In the present study, a wide range of 749 plasma metabolites, 62 urine biogenic amines, and 1,263 plasma proteins were analyzed in 10 healthy male volunteers measured repeatedly during 12 hours under tightly controlled conditions. Three variability components in relative concentration data are determined using linear mixed models: between (intersubject), time (intrasubject), and noise (intrasubject). Biomolecules such as 3-carboxy-4-methyl-5-propyl-2-furanpropanoate, platelet-derived growth factor C, and cathepsin D with low noise potentially detect changing conditions within a person. If also the between component is low, biomolecules can easier differentiate conditions between persons, for example cathepsin D, CD27 antigen, and prolylglycine. Variability over time does not necessarily inhibit translatability, but requires choosing sampling times carefully.

A Novel Orally Available Delta-5 Desaturase Inhibitor Prevents Atherosclerotic Lesions Accompanied by Changes in Fatty Acid Composition and Eicosanoid Production in ApoE Knockout Mice.

Delta-5 desaturase (D5D), encoded by fatty acid desaturase 1 (Fads1), is the rate-limiting enzyme for the conversion from dihomo-γ-linolenic acid (DGLA) to arachidonic acid (AA) in the ω-6 polyunsaturated fatty acid pathway. Several AA-derived eicosanoids (e.g., prostaglandins, thromboxanes, and leukotrienes) and DGLA-derived eicosanoids are reported to promote and/or prevent atherosclerosis progression through, at least in part, its proinflammatory or anti-inflammatory effects. To elucidate the effects of D5D inhibition by a D5D inhibitor on atherosclerosis, we generated a potent, orally available and selective D5D inhibitor, 2-(2,2,3,3,3-Pentafluoropropoxy)-3-[4-(2,2,2-trifluoroethoxy) phenyl]-5,7-dihydro-3H-pyrrolo[2,3-d]pyrimidine-4,6-dione, compound-326, and examined its effects on Western-diet fed ApoE knockout (KO) mice. Oral administration of compound-326 (3-10 mg/kg per day for 15 weeks) significantly inhibited the progression of atherosclerotic lesions in the aorta without affecting plasma total cholesterol and triglyceride levels. Compound-326 significantly decreased AA levels, while it increased DGLA levels in the liver and the blood accompanied by decreases in AA-derived eicosanoid production and increases in DGLA-derived eicosanoid production from the blood cells. We conclude that compound-326 prevents the progression of atherosclerosis in Western-diet fed ApoE KO mice by modulating a profile of eicosanoid production, suggesting that D5D inhibitors can be a novel remedy for preventing atherosclerosis and subsequent cardiovascular events. SIGNIFICANCE STATEMENT: This study shows a D5D-specific and orally available potent inhibitor provided the first evidence to support the concept that D5D inhibitors will be a novel remedy for preventing the progression of atherosclerosis.

Deoxysphingolipids and ether-linked diacylglycerols accumulate in the tissues of aged mice.

BACKGROUND: Senescence is a well-known risk factor for several diseases, such as neurodegenerative disorders. Therefore, studies exploring the mechanisms underlying aging are expected to guide the discovery of novel drug targets and biomarkers for these diseases. However, a comprehensive overview of the metabolic and lipidomic changes in healthy aging mammals is lacking. To understand the changes of metabolism with aging, especially lipid metabolism, we analyzed the metabolomes and lipidomes of the cerebral cortex, liver, femoral muscle, and epididymal fat in young and aged mice. RESULTS: Two-dimensional cluster analysis revealed clear separation between the metabolite profiles of the aged and young groups. Deoxydihydroceramide (doxDHCer), deoxyceramide (doxCer), and ether-linked diacylglycerol (DAG) levels were elevated during aging. CONCLUSION: This is the first report of age-related variations in deoxysphingolipid and ether-linked DAG levels in mice. DoxCer, doxDHCer, and ether-linked DAGs may be associated with senescence in mammalian tissues.

Inhibition of GCN2 sensitizes ASNS-low cancer cells to asparaginase by disrupting the amino acid response.

General control nonderepressible 2 (GCN2) plays a major role in the cellular response to amino acid limitation. Although maintenance of amino acid homeostasis is critical for tumor growth, the contribution of GCN2 to cancer cell survival and proliferation is poorly understood. In this study, we generated GCN2 inhibitors and demonstrated that inhibition of GCN2 sensitizes cancer cells with low basal-level expression of asparagine synthetase (ASNS) to the antileukemic agent l-asparaginase (ASNase) in vitro and in vivo. We first tested acute lymphoblastic leukemia (ALL) cells and showed that treatment with GCN2 inhibitors rendered ALL cells sensitive to ASNase by preventing the induction of ASNS, resulting in reduced levels of de novo protein synthesis. Comprehensive gene-expression profiling revealed that combined treatment with ASNase and GCN2 inhibitors induced the stress-activated MAPK pathway, thereby triggering apoptosis. By using cell-panel analyses, we also showed that acute myelogenous leukemia and pancreatic cancer cells were highly sensitive to the combined treatment. Notably, basal ASNS expression at protein levels was significantly correlated with sensitivity to combined treatment. These results provide mechanistic insights into the role of GCN2 in the amino acid response and a rationale for further investigation of GCN2 inhibitors for the treatment of cancer.

A Simple and Highly Sensitive Quantitation of Eicosanoids in Biological Samples Using Nano-flow Liquid Chromatography/Mass Spectrometry.

A simple sample preparation method for eicosanoid was developed by the combination of deproteinization and nanoLC-ESI-MS/MS. Eicosanoids are a group of bioactive lipid mediators, present in trace amounts in the body. Therefore, an analytical method for eicosanoids requires superior sensitivity. The method described in this report, which takes advantage of the highly sensitive power of nanoLC-ESI-MS/MS, enabled a simplification of the sample-preparation process. Eicosanoid extraction was performed just by homogenization in methanol with subsequent phospholipid removal, and then the liquid phase was directly subjected to nanoLC-ESI-MS/MS analysis without a condensation process. The quantitation range achieved 0.01 - 100 ng/mL for thromboxane B2, and 0.05 - 100 ng/mL for prostaglandin E2, prostaglandin D2, prostaglandin F2, leukotriene B4, 6-keto prostaglandin F1α and 11-dehydro thromboxane B2. Rat brain sample analyses demonstrated the feasibility of the quantification of those seven eicosanoids from biological samples.

Intratumor Heterogeneity in Primary Kidney Cancer Revealed by Metabolic Profiling of Multiple Spatially Separated Samples within Tumors.

Metabolic alteration constitutes a hallmark of cancer. Glycolysis and antioxidant pathways in kidney cancer are elevated, with frequent mutation of the VHL gene. Intratumor genetic heterogeneity has been recently demonstrated in kidney cancer. However, intratumor metabolic heterogeneity has not been investigated. Here, we used global metabolomics analysis and tissue slice tracer studies to demonstrate that different portions of a human primary kidney tumor possess different metabolic characteristics and drug sensitivity. Pyruvate levels were elevated and pyruvate metabolism was altered in some tumor sections. These observations indicated that pyruvate metabolism may constitute a possible vulnerability of kidney cancer; indeed, pyruvate stimulated the growth of primary kidney cancer cells and pharmacological inhibition of pyruvate transporters slowed the growth of patient-derived kidney tumors in mice. These findings deepen our understanding of the intratumor metabolic heterogeneity of kidney cancer and may inform novel therapeutic approaches in human kidney cancer.

Succinate dehydrogenase B-deficient cancer cells are highly sensitive to bromodomain and extra-terminal inhibitors.

Mutations in succinate dehydrogenase B (SDHB) gene are frequently observed in several tumors and associated with poor prognosis in these tumors. Therefore, drugs effective for SDHB-deficient tumors could fulfill an unmet medical need. In addition, such drugs would have an advantage in that selection of patients with SDHB-mutant cancer could increase the probability of success in clinical trials. Currently, however, the characteristics of SDHB-deficient cancers are not completely understood. Here, we established SDHB knockout cancer cell lines from human colon cancer HCT116 cells using the clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 knockout system, and clarified its metabolic characteristics.In the SDHB knockout cells, succinate was accumulated and fumarate was decreased. The oxygen consumption rate was decreased while the extracellular acidification rate was increased in the SDHB knockout cells. Accordingly, an enhanced glycolysis pathway in the SDHB knockout cells was demonstrated by metabolomics analysis. Tracer experiments showed bidirectional metabolic flow in the tricarboxylic acid (TCA) cycle, possibly to maintain the necessary amounts of metabolites in the SDHB knockout cells. The proliferation of SDHB knockout cells was suppressed by a glycolysis inhibitor but not by a mitochondrial inhibitor. Additionally, partial dependence on glutaminolysis was observed in the SDHB knockout cells. Compound screening revealed that a bromodomain and extra-terminal (BET) inhibitor, which downregulated c-Myc, suppressed the growth of the SDHB knockout cells more potently than that of control cells. These findings provide an understanding of the metabolic characteristics of SDHB-deficient cancer and its vulnerabilities, which may lead to new therapeutic options.

A novel and selective melanin-concentrating hormone receptor 1 antagonist ameliorates obesity and hepatic steatosis in diet-induced obese rodent models.

Melanin-concentrating hormone (MCH), a cyclic neuropeptide expressed predominantly in the lateral hypothalamus, plays an important role in the control of feeding behavior and energy homeostasis. Mice lacking MCH or MCH1 receptor are resistant to diet-induced obesity (DIO) and MCH1 receptor antagonists show potent anti-obesity effects in preclinical studies, indicating that MCH1 receptor is a promising target for anti-obesity drugs. Moreover, recent studies have suggested the potential of MCH1 receptor antagonists for treatment of non-alcoholic fatty liver disease (NAFLD). In the present study, we show the anti-obesity and anti-hepatosteatosis effect of our novel MCH1 receptor antagonist, Compound A. Repeated oral administration of Compound A resulted in dose-dependent body weight reduction and had an anorectic effect in DIO mice. The body weight lowering effect of Compound A was more potent than that of pair-feeding. Compound A also reduced lipid content and the expression level of lipogenesis-, inflammation-, and fibrosis-related genes in the liver of DIO mice. Conversely, intracerebroventricular infusion of MCH caused induction of hepatic steatosis as well as increase in body weight in high-fat diet-fed wild type mice, but not MCH1 receptor knockout mice. The pair-feeding study revealed the MCH-MCH1 receptor system affects hepatic steatosis through a mechanism that is independent of body weight change. Metabolome analysis demonstrated that Compound A upregulated lipid metabolism-related molecules, such as acylcarnitines and cardiolipins, in the liver. These findings suggest that our novel MCH1 receptor antagonist, Compound A, exerts its beneficial therapeutic effect on NAFLD and obesity through a central MCH-MCH1 receptor pathway.

Associations of number of teeth with risks for all-cause mortality and cause-specific mortality in middle-aged and elderly men in the northern part of Japan: the Iwate-KENCO study.

OBJECTIVES: The objective of this study was to determine the associations of number of teeth with all-cause mortality and cause-specific mortality among middle-aged and elderly Japanese men. METHODS: A total of 7779 men aged 40-79 years who were free from cardiovascular disease (CVD) were followed up prospectively for 5.6 years. Participants were categorized into four groups (no teeth, 1-9 teeth, 10-19 teeth, and ≥20 teeth) by a self-administered questionnaire. Using Cox's proportional hazard model, multivariate hazard ratios (HRs) and 95% confidence intervals (CIs) for mortality from all causes, CVD, cancer, and noncancer, non-CVD according to number of teeth were estimated with adjustments for age, body mass index, systolic blood pressure, total- and HDL-cholesterol, HbA1c, current smoking, current alcohol drinking, and low level of education. RESULTS: The numbers (proportions) of participants with no teeth, 1-9 teeth, 10-19 teeth, and ≥20 teeth were 1613 (20.7%), 1650 (21.2%), 1721 (22.1%), and 2795 (35.9%), respectively. During follow-up, a total of 455 deaths (including 175 deaths from cancer, 98 deaths from CVD, and 130 deaths from noncancer, non-CVD) were recorded. In total participants, an inverse relationship between number of teeth and all-cause mortality was found (P for trend = 0.049). Among men aged 40-64 years, inverse relationships were also found in risks for mortality from all causes, CVD, and cancer: multivariate-adjusted HRs (95% CI) for all-cause mortality in men with no teeth, 1-9 teeth, and 10-19 teeth relative to men with ≥20 teeth were 2.75 (1.37-5.49), 1.89 (0.99-3.63), and 1.94 (1.09-3.43), respectively. However, there were no associations of number of teeth with all-cause mortality and cause-specific mortality among men aged 65-79 years. CONCLUSIONS: The number of teeth is an important predictive factor for mortality among middle-aged Japanese men.

Factors related to tooth loss among community-dwelling middle-aged and elderly Japanese men.

BACKGROUND: Using data from a large-scale community-based Japanese population, we attempted to identify factors associated with tooth loss in middle-aged and elderly men. METHODS: A total of 8352 men aged 40 to 79 years who lived in the north of the main island of Japan and underwent health checkups were enrolled between 2002 and 2005. Number of teeth was assessed by the question, "How many teeth do you have (0, 1-9, 10-19, or ≥20)?". On the basis of the answer to this question, participants were classified into 2 groups (≤19 teeth or ≥20 teeth). Using multivariate logistic regression, factors related to having 19 or fewer teeth were estimated. RESULTS: The numbers (percentages) of participants who had 0, 1 to 9, 10 to 19, and 20 or more teeth were 1764 (21.1%), 1779 (21.3%), 1836 (22.0%), and 2973 (35.6%), respectively. Among the participants overall and those aged 65 to 79 years, having 19 or fewer teeth was significantly associated with older age, smoking status (current smoking and ex-smoking), and low education level. In addition, men with 19 or fewer teeth were more likely to have a low body mass index and low serum albumin level and less likely to be current alcohol drinkers. Among men aged 40 to 64 years, but not men aged 65 to 79 years, those with 19 or fewer teeth were more likely to have a low serum high-density lipoprotein cholesterol level and high glycosylated hemoglobin (HbA1c) level. CONCLUSIONS: Smoking, low education level, and poor nutritional status were associated with tooth loss among middle-aged and elderly Japanese men.

Efficient expression of haloarchaeal nucleoside diphosphate kinase via strong porin promoter in moderately halophilic bacteria.

Enzymes from extremely halophilic archaea require high concentration of salts for their proper folding and consequently are expressed as an unfolded and inactive form in Escherichia coli. Moderate halophile, which accumulates protein stabilizers, i.e., compatible solutes, is an attractive host cell for the recombinant production of heterologous proteins, since such protein stabilizers may help folding of expressed proteins. Here, we succeeded in efficient expression and purification to homogeneity of recombinant haloarchaeal nucleoside diphosphate kinase (HsNDK) in moderate halophile using newly isolated strong porin promoter.

Correlation of cariogenic bacteria and dental caries in adults.

Many studies suggest that mutans streptococci (MS), Lactobacillus (LB), and salivary buffering capacity are important risk factors for dental caries. However, target populations for most studies were children. In adult patients, the same risk factors affect the number of fillings or prostheses or secondary caries. It is therefore important to investigate these risk factors as predictors of caries in adults. In the present study, we evaluated the oral conditions of adult subjects at private dental offices using bite-wing radiographs. Detection of salivary LB level using Dentocult LB had a statistically significant correlation with the number of flat surface caries and approximal caries (P < 0.001). Detection of salivary MS level using Dentocult MS and salivary buffering capacity did not predict dental caries. Thus, detection of salivary LB level using Dentocult LB may be a useful tool for detecting approximal and secondary caries.