Title: Contribution of intestinal dipeptidyl peptidase-4 inhibition for incretin-dependent improved glucose tolerance in mice Yamashita S, Kawakami Y, Sato H, Sugitani S, Goto M, Kato N Ref: European Journal of Pharmacology, :172521, 2019 : PubMed
Dipeptidyl peptidase-4 (DPP-4) inhibitors prevent the degradation of glucagon-like peptide-1 (GLP-1) and improve glycemic control. The GLP-1 insulinotropic effect involves a pathway through vagus nerve GLP-1 receptors in the gut, in addition to a direct effect on the pancreas. Therefore, this study verified whether DPP-4 inhibition in the gut contributed to the improvement of glycemic control. Anagliptin, a DPP-4 inhibitor, was administered orally or subcutaneously (with or without passing through the gastrointestinal tract, respectively) to mice. The association between blood glucose suppression following oral glucose challenge and DPP-4 inhibition in the small intestine and plasma was assessed. Oral administration of anagliptin (0.03-0.3mg/kg) in normal mice significantly suppressed blood glucose, which was associated with an increase in insulin secretion at a dose of >/=0.1mg/kg (P<0.05). Subcutaneous administration of anagliptin (0.01-0.1mg/kg) produced similar results. However, plasma DPP-4 inhibition following oral administration was weaker than that following subcutaneous administration; blood glucose suppression was significantly correlated with small intestinal DPP-4 inhibition (r=0.949, P<0.01), but not with plasma DPP-4 inhibition. Additionally, similar results were observed in a type 2 diabetes model (r=0.975, P<0.001). Thus, these results demonstrated that an improvement in glycemic control was dependent upon small intestinal DPP-4 inhibition. As these effects were accompanied by the elevation of intact GLP-1 in the portal, this suggests that improvement in glucose tolerance after anagliptin treatment might be related to an increase in GLP-1 receptor signaling in the small intestine and portal vein.
OBJECTIVES: Brain acetylcholine is decreased even in patients with cognitively preserved Parkinson's disease (PD). We investigated whether early and long-term use of donepezil prevents psychosis in non-demented PD patients. METHODS: A double-blinded, placebo-controlled trial was conducted. A total of 145 non-demented PD patients were randomly assigned to receive 5 mg/day donepezil (n=72) or placebo (n=73) for 96 weeks. Medications for PD were not restricted, but antipsychotic drugs were not permitted throughout the study. The primary outcome measure was survival time to psychosis that was predefined by Parkinson's Psychosis Questionnaire (PPQ) B score >/=2 or C score >/=2. Secondary outcome measures included psychosis developing within 48 weeks, total PPQ score, Mini-Mental State Examination (MMSE), Wechsler Memory Scale (WMS) and subgroup analysis by apolipoprotein epsilon4 genotyping. RESULTS: Kaplan-Meier curves for psychosis development were very similar between the two groups, and the Cox proportional hazard model revealed an adjusted HR of 0.87 (95%CI 0.48 to 1.60). The changes in MMSE and WMS-1 (auditory memory) were significantly better with donepezil than in placebo. In the subgroup analysis, donepezil provided an HR of 0.31 (0.11-0.86) against psychosis in 48 weeks for apolipoprotein epsilon4 non-carriers. CONCLUSIONS: Although donepezil provided beneficial effects on PPQ, MMSE and auditory WMS score changes in 2 years, it had no prophylactic effect on development of psychosis in PD. Apolipoprotein epsilon4 may suppress the antipsychotic effect of donepezil. TRIAL REGISTRATION NUMBER: UMIN000005403.
AIMS/INTRODUCTION: Dipeptidyl peptidase-4 inhibitors are used for treatment of patients with type 2 diabetes. In addition to glycemic control, these agents showed beneficial effects on lipid metabolism in clinical trials. However, the mechanism underlying the lipid-lowering effect of dipeptidyl peptidase-4 inhibitors remains unclear. Here, we investigated the lipid-lowering efficacy of anagliptin in a hyperlipidemic animal model, and examined the mechanism of action. MATERIALS AND METHODS: Male low-density lipoprotein receptor-deficient mice were administered 0.3% anagliptin in their diet. Plasma lipid levels were assayed and lipoprotein profile was analyzed using high-performance liquid chromatography. Hepatic gene expression was examined by deoxyribonucleic acid microarray and quantitative polymerase chain reaction analyses. Sterol regulatory element-binding protein transactivation assay was carried out in vitro. RESULTS: Anagliptin treatment significantly decreased the plasma total cholesterol (14% reduction, P < 0.01) and triglyceride levels (27% reduction, P < 0.01). Both low-density lipoprotein cholesterol and very low-density lipoprotein cholesterol were also decreased significantly by anagliptin treatment. Sterol regulatory element-binding protein-2 messenger ribonucleic acid expression level was significantly decreased at night in anagliptin-treated mice (15% reduction, P < 0.05). Anagliptin significantly suppressed sterol regulatory element-binding protein activity in HepG2 cells (21% decrease, P < 0.001). CONCLUSIONS: The results presented here showed that the dipeptidyl peptidase-4 inhibitor, anagliptin, exhibited a lipid-lowering effect in a hyperlipidemic animal model, and suggested that the downregulation of hepatic lipid synthesis was involved in the effect. Anagliptin might have beneficial effects on lipid metabolism in addition to a glucose-lowering effect.
Title: Anagliptin, a dipeptidyl peptidase-4 inhibitor, decreases macrophage infiltration and suppresses atherosclerosis in aortic and coronary arteries in cholesterol-fed rabbits Hirano T, Yamashita S, Takahashi M, Hashimoto H, Mori Y, Goto M Ref: Metabolism, 65:893, 2016 : PubMed
INTRODUCTION: Several studies have demonstrated suppression of aortic atherosclerosis by dipeptidyl peptidase-4 (DPP-4) inhibitors in hypercholesterolemic mice. However, it remains unknown whether DPP-4 inhibitors also exert anti-atherogenic effects in coronary arteries. We examined the effect of anagliptin, a DPP-4 inhibitor, on atherosclerosis development in the aorta and coronary arteries in a high-cholesterol diet-fed rabbits. METHODS: Japanese white rabbits were fed either normal chow (n=8) or a diet containing 0.5% cholesterol (n=34) for 14weeks. Cholesterol-fed rabbits were given 0.3% anagliptin or not in drinking water (each n=16 and 18) for 12weeks. RESULTS: Dietary cholesterol intake markedly increased serum total cholesterol (TC) levels (1464+/-150mg/dL, mean+/-SE), and the most striking increase was observed among the major lipoproteins in very low-density lipoprotein (VLDL) as determined by high-performance liquid chromatography. No significant changes were observed in body weight, water intake, hemoglobin A1c, or glucose response to intravenous glucose loading following anagliptin administration. Anagliptin decreased TC and VLDL-cholesterol as well as cholesterol absorption markers sitosterol and campesterol slightly, although not significantly. Serum DPP-4 activity was suppressed by 82%, and active glucagon-like peptide-1 and glucose-dependent insulinotropic polypeptide levels were increased 2- to 3-fold by anagliptin treatment. Severe hypercholesterolemia resulted in the development of atherosclerosis in the aorta, and the ratio of atherosclerotic lesions to the total aortic surface area was 22+/-2%. Anagliptin suppressed the lesion ratio to 9+/-2% (p<0.001). Atherosclerotic lesions were clearly observed in the coronary arteries, where the mean intima-media area was enlarged, and intimal formation was developed. Anagliptin treatment attenuated the intima-media area and the intimal area by 43%. Alpha-smooth muscle actin-positive and macrophage-positive areas in the coronary arteries were suppressed by 66 and 75%, respectively, after anagliptin treatment. The aortic lesion ratio and the coronary intima area were correlated with each other (r=0.506, p<0.01), and each lesion correlated with TC in the whole cholesterol-fed rabbits. Gene expression of the proinflammatory cytokines tumor necrosis factor-alpha and interleukin-6 in the carotid arteries was markedly reduced by approximately 90%, and vascular DPP-4 activity was reduced by 66% after anagliptin treatment. CONCLUSIONS: We demonstrated for the first time that a DPP-4 inhibitor can substantially suppress plaque formation in coronary arteries with a marked reduction in macrophage accumulation likely via its anti-inflammatory properties.
Title: Add-on therapy with anagliptin in Japanese patients with type-2 diabetes mellitus treated with metformin and miglitol can maintain higher concentrations of biologically active GLP-1/total GIP and a lower concentration of leptin Osonoi T, Saito M, Hariya N, Goto M, Mochizuki K Ref: Peptides, 86:118, 2016 : PubMed
Metformin, alpha-glucosidase inhibitors (alpha-GIs), and dipeptidyl peptidase 4 inhibitors (DPP-4Is) reduce hyperglycemia without excessive insulin secretion, and enhance postprandial plasma concentration of glucagon-like peptide-1 (GLP-1) in type-2 diabetes mellitus (T2DM) patients. We assessed add-on therapeutic effects of DPP-4I anagliptin in Japanese T2DM patients treated with metformin, an alpha-GI miglitol, or both drugs on postprandial responses of GLP-1 and glucose-dependent insulinotropic polypeptide (GIP), and on plasma concentration of the appetite-suppressing hormone leptin. Forty-two Japanese T2DM patients with inadequately controlled disease (HbA1c: 6.5%-8.0%) treated with metformin (n=14), miglitol (n=14) or a combination of the two drugs (n=14) received additional treatment with anagliptin (100mg, p.o., b.i.d.) for 52 weeks. We assessed glycemic control, postprandial responses of GLP-1 and glucose-dependent insulinotropic polypeptide (GIP), and on plasma concentration of leptin in those patients. Add-on therapy with anagliptin for 52 weeks improved glycemic control and increased the area under the curve of biologically active GLP-1 concentration without altering obesity indicators. Total GIP concentration at 52 weeks was reduced by add-on therapy in groups treated with miglitol compared with those treated with metformin. Add-on therapy reduced leptin concentrations. Add-on therapy with anagliptin in Japanese T2DM patients treated with metformin and miglitol for 52 weeks improved glycemic control and enhanced postprandial concentrations of active GLP-1/total GIP, and reduce the leptin concentration.
AIMS/HYPOTHESIS: Recently, incretin-related agents have been reported to attenuate insulin resistance in animal models, although the underlying mechanisms remain unclear. In this study, we investigated whether anagliptin, the dipeptidyl peptidase 4 (DPP-4) inhibitor, attenuates skeletal muscle insulin resistance through endothelial nitric oxide synthase (eNOS) activation in the endothelial cells. We used endothelium-specific Irs2-knockout (ETIrs2KO) mice, which show skeletal muscle insulin resistance resulting from a reduction of insulin-induced skeletal muscle capillary recruitment as a consequence of impaired eNOS activation. METHODS: In vivo, 8-week-old male ETIrs2KO mice were fed regular chow with or without 0.3% (wt/wt) DPP-4 inhibitor for 8 weeks to assess capillary recruitment and glucose uptake by the skeletal muscle. In vitro, human coronary arterial endothelial cells (HCAECs) were used to explore the effect of glucagon-like peptide 1 (GLP-1) on eNOS activity. RESULTS: Treatment with anagliptin ameliorated the impaired insulin-induced increase in capillary blood volume, interstitial insulin concentration and skeletal muscle glucose uptake in ETIrs2KO mice. This improvement in insulin-induced glucose uptake was almost completely abrogated by the GLP-1 receptor (GLP-1R) antagonist exendin-(9-39). Moreover, the increase in capillary blood volume with anagliptin treatment was also completely inhibited by the NOS inhibitor. GLP-1 augmented eNOS phosphorylation in HCAECs, with the effect completely disappearing after exposure to the protein kinase A (PKA) inhibitor H89. These data suggest that anagliptin treatment enhances insulin-induced capillary recruitment and interstitial insulin concentrations, resulting in improved skeletal muscle glucose uptake by directly acting on the endothelial cells via NO- and GLP-1-dependent mechanisms in vivo. CONCLUSIONS/INTERPRETATION: Anagliptin may be a promising agent to ameliorate skeletal muscle insulin resistance in obese patients with type 2 diabetes.
Awamori is a traditional distilled beverage made from steamed Thai-Indica rice in Okinawa, Japan. For brewing the liquor, two microbes, local kuro (black) koji mold Aspergillus luchuensis and awamori yeast Saccharomyces cerevisiae are involved. In contrast, that yeasts are used for ethanol fermentation throughout the world, a characteristic of Japanese fermentation industries is the use of Aspergillus molds as a source of enzymes for the maceration and saccharification of raw materials. Here we report the draft genome of a kuro (black) koji mold, A. luchuensis NBRC 4314 (RIB 2604). The total length of nonredundant sequences was nearly 34.7 Mb, comprising approximately 2,300 contigs with 16 telomere-like sequences. In total, 11,691 genes were predicted to encode proteins. Most of the housekeeping genes, such as transcription factors and N-and O-glycosylation system, were conserved with respect to Aspergillus niger and Aspergillus oryzae An alternative oxidase and acid-stable alpha-amylase regarding citric acid production and fermentation at a low pH as well as a unique glutamic peptidase were also found in the genome. Furthermore, key biosynthetic gene clusters of ochratoxin A and fumonisin B were absent when compared with A. niger genome, showing the safety of A. luchuensis for food and beverage production. This genome information will facilitate not only comparative genomics with industrial kuro-koji molds, but also molecular breeding of the molds in improvements of awamori fermentation.
The filamentous fungus Aspergillus kawachii has traditionally been used for brewing the Japanese distilled spirit shochu. A. kawachii characteristically hyperproduces citric acid and a variety of polysaccharide glycoside hydrolases. Here the genome sequence of A. kawachii IFO 4308 was determined and annotated. Analysis of the sequence may provide insight into the properties of this fungus that make it superior for use in shochu production, leading to the further development of A. kawachii for industrial applications.
Acetylcholinesterase and butyrylcholinesterase (BChE) activities in blood are widely used as the biomarkers for organophosphorus insecticide (OP) exposure. In the present study, we conducted a cross-sectional study to evaluate plasma beta-glucuronidase (BG), a sensitive biomarker candidate for OP exposure, BChE activities and urinary dialkyl phosphates (DAPs), OP metabolites. We assessed the relationship between these biomarker levels in the following groups: 32 controls (control), 21 pest control operators and their co-workers who had not sprayed OPs within 3 days prior to sample collection (PCO1), and 21 pest control operators who sprayed OPs within those 3 days (PCO2). Logarithmically transformed age-adjusted means of DAPs were 3.88, 5.62 and 6.45 nmol/g creatinine for control, PCO1 and PCO2, respectively (P<0.001 for difference, P<0.001 for trend). Logarithmically transformed age-adjusted means of BG were 1.40, 1.52 and 1.85 micromol/L/h for control, PCO1 and PCO2, respectively. BG activity, but not BChE, was increased according to their OP exposure level (P=0.038 for difference, P=0.026 for trend). It was concluded that plasma BG activity is more sensitive biomarker as well as urinary OP metabolites than BChE for low-level exposure in humans.
Title: Emergence of two types of nondechlorinating variants in the tetrachloroethene-halorespiring Desulfitobacterium sp. strain Y51 Futagami T, Tsuboi Y, Suyama A, Goto M, Furukawa K Ref: Applied Microbiology & Biotechnology, 70:720, 2006 : PubMed
Desulfitobacterium sp. strain Y51 exhibits a strong dechlorinating activity for tetrachloroethene (PCE), converting it to cis-1,2-dichloroethene via trichloroethene by the action of the PceA reductive dehalogenase (encoded by pceA). The gene organization around the pceA gene cluster was determined to be in the following order: orf4, orf3, ISDesp1, pceA-B-C-T-mcpA, and ISDesp2, where the pceA gene cluster is surrounded by two nearly identical copies of the ISDesp insertion sequence. Serial subculture of strain Y51 gave rise to variants that abolished the PCE-dechlorination activity. Southern hybridization analysis revealed two types of variants termed small deletion (SD) and large deletion (LD). The characterization of both variants revealed a genetic rearrangement around the pceAB gene cluster. In variant SD, ISDesp1 comprised of 1,572 bp was deleted, which includes the tnpAa encoding IS256 family transposase and unknown orf1. The ISDesp1 contained the inverted terminal repeat sequence and a -35 promoter stretch just upstream of the pceA gene, indicating that this IS element is involved in the formation of the variant SD. Loss of the pceA transcription changed the variant SD to the PCE-nondechlorinating phenotype. The variant LD lost the 6.5-kb region, including one copy of ISDesp and the pceABCT-mcpA gene cluster, confirming that the homologous recombination is associated with the emergence of this variant.
Title: Poly(ethylene glycol)-lipase complexes that are highly active and enantioselective in ionic liquids Maruyama T, Yamamura H, Kotani T, Kamiya N, Goto M Ref: Org Biomol Chem, 2:1239, 2004 : PubMed
Lipase-catalyzed alcoholysis between vinyl acetate and 2-phenyl-1-propanol was investigated in dialkylimidazolium-based ionic liquids. Although native lipase powder exhibited very low activity in an ionic liquid, forming a poly(ethylene glycol)(PEG)-lipase complex improved the lipase activity in the ionic liquid. The activity of the PEG-lipase complex was higher in ionic liquids than in common organic solvents (n-hexane, isooctane and dimethylsulfoxide). Fluorescence measurements using 4-aminophthalimide revealed that the ionic liquids were more hydrophilic than the organic solvents used for non-aqueous enzymology. A kinetic study of lipase-catalyzed alcoholysis in an ionic liquid ([Bmim][PF6]) revealed that the Michaelis constant (Km) for 2-phenyl-1-propanol in the ionic liquid was half that in n-hexane, suggesting that the ionic liquid stabilized the enzyme-substrate complex. Finally, we carried out enantioselective alcoholysis of 1-phenylethanol in ionic liquids employing the PEG-lipase complex, and obtained high enantioselectivity, comparable to that in n-hexane.
Title: Transport of organic acids through a supported liquid membrane driven by lipase-catalyzed reactions Miyako E, Maruyama T, Kamiya N, Goto M Ref: J Biosci Bioeng, 96:370, 2003 : PubMed
We have developed a lipase-facilitated supported liquid membrane. Lipase-catalyzed reactions were coupled with a supported liquid membrane (SLM) to transport organic acids through the SLM. We succeeded in the rational transport of organic acids through the SLM using lipase-catalyzed reactions and observed that there were differences in the transport behavior of various organic acids due to the substrate specificity of lipase. Subsequently, various parameters, such as the alcohol concentration in the feed phase, the pH in each aqueous phase, an organic solvent in the SLM, and the kind of lipase, were investigated. We found that the optimum conditions were 65 vol% alcohol concentration, pH 6.3 in each aqueous phase, isooctane as the liquid membrane phase and Candida rugosa lipase as the esterification biocatalyst.
Using an animal model of forebrain ischemia in spontaneously hypertensive rats (SHR) by 3-h bilateral carotid occlusion, and various indices of the cerebral cholinergic system were assessed for periods up to 24 weeks. The lesions observed histologically in the hippocampus of SHR 2 weeks after ischemia were less severe than those in the frontal cortex. Marked elevation of acetylcholine concentration was transiently observed in the frontal cortex, hippocampus and thalamus + midbrain at 2 weeks, and in the striatum at 1-4 weeks after ischemia. Choline acetyltransferase activity remained unchanged in all regions throughout the experimental period except for a minimal decrease in the frontal cortex at 4 weeks. Choline esterase (ChE) activity was slightly decreased in the frontal cortex at 2-4 weeks after ischemia but recovered by 8 weeks. A decrease in the hippocampus was seen at 8 weeks. The B(max) for the M1-receptor was significantly reduced by 2 weeks in the frontal cortex and by 4 weeks in the hippocampus. Low B(max) values in both regions persisted through week 24. These delayed hippocampal changes in the ChE activity and M1-receptor in SHR were similar to those of the very much delayed changes in M1-receptor previously reported in the gerbil model for transient ischemia. In contrast, Wistar-Kyoto rats (WKY), used as normotensive controls, exhibited no histological or biochemical changes for up to 24 weeks. The difference between SHR and WKY may depend on the more severe cerebral blood flow depletion during carotid ligation in the former. The chronic state of SHR after the transient ischemia may be a useful pathophysiological model for human cerebral infarctions with hypertension.
