Orlistat, an anti-obesity agent, inhibits the metabolism and absorption of dietary fat by inactivating pancreatic lipase in the gut. The effect of orlistat on the gut microbiota of Japanese individuals with obesity is unknown. This study aimed to explore the effects of orlistat on the gut microbiota and fatty acid metabolism of Japanese individuals with obesity. Fourteen subjects with visceral fat obesity (waist circumference <=85 cm) took orlistat orally at a dose of 60 mg, 3 times a day for 8 weeks. Body weight; waist circumference; visceral fat area; levels of short-chain fatty acids, gut microbiota, fatty acid metabolites in the feces, and gastrointestinal hormones; and adverse events were evaluated. Body weight, waist circumference, and blood leptin concentrations were significantly lower after orlistat treatment (mean +/- standard deviation, 77.8 +/- 9.1 kg; 91.9 +/- 8.7 cm; and 4546 +/- 3211 pg/mL, respectively) compared with before treatment (79.4 +/- 9.0 kg; 94.4 +/- 8.0 cm; and 5881 +/- 3526 pg/mL, respectively). Significant increases in fecal levels of fatty acid metabolites (10-hydroxy-cis-12-octadecenoic acid, 10-oxo-cis-12-octadecenoic acid, and 10-oxo-trans-11-octadecenoic acid) were detected. Meanwhile, no significant changes were found in abdominal computed tomography parameters, blood marker levels, or short-chain fatty acid levels in the feces. Gut microbiota analysis revealed that some study subjects had decreased abundance of Firmicutes, increased abundance of Bacteroidetes, and increased alpha-diversity indices (Chao1 and ACE) after 8 weeks of treatment. The levels of Lactobacillus genus and Lactobacillus gasseri were significantly higher after 8 weeks of treatment. None of the subjects discontinued treatment or experienced severe adverse events. This study suggested that orlistat might alter gut microbiota composition and affect the body through fatty acid metabolites produced by the modified gut bacteria.
BACKGROUND: Nonalcoholic fatty liver disease (NAFLD) refers to hepatic steatosis caused by something other than alcoholic liver injury, and often occurs after gastrointestinal surgeries such as pancreatoduodenectomy and gastrectomy. This study aimed to identify the risk factors for NAFLD after gastrectomy for gastric cancer. METHODS: A total of 721 patients who underwent gastrectomy for gastric cancer and plane abdominal computed tomography (CT) preoperatively and 1 year after surgery were included in this study. NAFLD was defined as having a mean hepatic CT attenuation value of < 40 Hounsfield units. We retrospectively examined the relationship between the onset of NAFLD and clinicopathological findings to identify the risk factors associated with the development of NAFLD after gastrectomy. RESULTS: The incidence of postoperative NAFLD was 4.85% (35/721). The univariate analysis identified the following factors as being significantly associated with the incidence of NAFLD: age, preoperative BMI >/= 25 kg/m(2), tumor depth of pT3
Title: Carboxypeptidase Y activity and maintenance is modulated by a large helical structure Makino M, Sahara T, Morita N, Ueno H Ref: FEBS Open Bio, 9:1337, 2019 : PubMed
Yeast carboxypeptidase Y (CPY) is a serine protease with broad substrate specificity. Structurally, CPY belongs to the alpha/beta hydrolase fold family and contains characteristic large helices, termed the V-shape helix, above the active site cavity. Four intramolecular disulfide bonds are located in and around the V-shape helix. In this study, mutant CPYs were constructed in which one of these disulfide bonds was disrupted. Mutants lacking the C193-C207 bond located at the beginning of the V-shape helix aggregated easily, while mutants lacking the C262-C268 bond located at the end of the V-shape helix displayed decreased hydrolytic activity. The results indicate that the V-shape helix is involved in CPY catalysis and in maintenance of its conformation.
Trelagliptin (SYR-472), a novel dipeptidyl peptidase-4 inhibitor, shows sustained efficacy by once-weekly dosing in type 2 diabetes patients. In this study, we characterized in vitro properties of trelagliptin, which exhibited approximately 4- and 12-fold more potent inhibition against human dipeptidyl peptidase-4 than alogliptin and sitagliptin, respectively, and >10,000-fold selectivity over related proteases including dipeptidyl peptidase-8 and dipeptidyl peptidase-9. Kinetic analysis revealed reversible, competitive and slow-binding inhibition of dipeptidyl peptidase-4 by trelagliptin (t1/2 for dissociation approximately 30 minutes). X-ray diffraction data indicated a non-covalent interaction between dipeptidyl peptidase and trelagliptin. Taken together, potent dipeptidyl peptidase inhibition may partially contribute to sustained efficacy of trelagliptin.
Title: Increased proteolytic susceptibility of carboxypeptidase Y caused by modification of the disulfide zipper Maki T, Kozawa H, Mima J, Ueno H, Hayashi R Ref: Biosci Biotechnol Biochem, 66:1393, 2002 : PubMed
To investigate the structural importance of a "disulfide zipper" motif of carboxypeptidase Y, disulfide-deficient mutant enzymes were expressed in two strains of Saccharomyces cerevisiae. The mutant enzymes were rapidly degraded into fragments by intracellular proteases. Thus, it is concluded that the disulfide zipper is essential in maintaining the structural integrity of CPase Y against proteolytic susceptibility.
Title: Amphipathic property of free thiol group contributes to an increase in the catalytic efficiency of carboxypeptidase Y Mima J, Jung G, Onizuka T, Ueno H, Hayashi R Ref: European Journal of Biochemistry, 269:3220, 2002 : PubMed
Cys341 of carboxypeptidase Y, which constitutes one side of the solvent-accessible surface of the S1 binding pocket, was replaced with Gly, Ser, Asp, Val, Phe or His by site-directed mutagenesis. Kinetic analysis, using Cbz-dipeptide substrates, revealed that polar amino acids at the 341 position increased K(m) whereas hydrophobic amino acids in this position tended to decrease K(m). This suggests the involvement of Cys341 in the formation of the Michaelis complex in which Cys341 favors the formation of hydrophobic interactions with the P1 side chain of the substrate as well as with residues comprising the surface of the S1 binding pocket. Furthermore, C341G and C341S mutants had significantly higher k(cat) values with substrates containing the hydrophobic P1 side chain than C341V or C341F. This indicates that the nonhydrophobic property conferred by Gly or Ser gives flexibility or instability to the S1 pocket, which contributes to the increased k(cat) values of C341G or C341S. The results suggest that Cys341 may interact with His397 during catalysis. Therefore, we propose a dual role for Cys341: (a) its hydrophobicity allows it to participate in the formation of the Michaelis complex with hydrophobic substrates, where it maintains an unfavorable steric constraint in the S1 subsite; (b) its interaction with the imidazole ring of His397 contributes to the rate enhancement by stabilizing the tetrahedral intermediate in the transition state.
Title: Carboxypeptidase Y: structural basis for protein sorting and catalytic triad Jung G, Ueno H, Hayashi R Ref: J Biochem, 126:1, 1999 : PubMed
A yeast vacuolar protease, carboxypeptidase Y (CPY), is known to be involved in the C-terminal processing of peptides and proteins; however, its real function remains unclear. The CPY biosynthetic pathway has been used as a model system for protein sorting in eukaryotes. CPY is synthesized as a prepro-form that travels through the ER and Golgi to its final destination in vacuoles. In the course of studies on the transport mechanism of CPY, various post-translational events have been identified, e.g. carbohydrate modification and cleavage of the pre-segments. In addition, sorting signals and various sorting vehicles, similar to those found in higher eukaryotic cells, have been found. The catalytic triad in the active site of CPY makes this enzyme a serine protease. A unique feature distinguishing CPY from other serine proteases is its wide pH optimum, in particular its high activity at acidic pH. Several structural properties which might contribute to this unique feature exist such as a conserved free cysteine residue in the S1 substrate binding pocket, a recognition site for a C-terminal carboxyl group, and a disulfide zipper motif. The structural bases in CPY functions are discussed in this article.
Title: Identification of the catalytic histidine residue participating in the charge-relay system of carboxypeptidase Y Jung G, Ueno H, Hayashi R, Liao TH Ref: Protein Science, 4:2433, 1995 : PubMed
The essential histidine residue of carboxypeptidase Y (CPY) was modified by a site-specific reagent, a chloromethylketone derivative of benzyloxycarbonyl-L-phenylalanine. The single modified histidine residue was converted to N tau-carboxy-methyl histidine (cmHis) upon performic acid oxidation. A peptide containing cmHis was isolated from the tryptic-thermolytic digest. Based on the amino acid composition and sequence analysis, the peptide is shown to be Val-Phe-Asp-Gly-Gly-cmHis-MetO2-Val-Pro, which was derived from CPY cleaved by trypsin at Arg 391 and thermolysin at Phe 401, and thus His 397 was modified. This histidine residue has been implicated previously by X-ray analysis to participate in the charge-relay system of CPY.
Title: Ultrastructural demonstration of acetylcholinesterase activity of motor endplates via osmiophilic diazothioethers Bergman RA, Ueno H, Morizono Y, Hanker JS, Seligman AM Ref: Histochemie, 11:1, 1967 : PubMed
