Mikiko Kishi

Thioesterase activity and subcellular localization of acylprotein thioesterase 1/lysophospholipase 1

Acylprotein thioesterase 1 (APT1), also known as lysophospholipase 1, is an important enzyme responsible for depalmitoylation of palmitoyl proteins. To clarify the substrate selectivity and the intracellular function of APT1, we performed kinetic analyses and competition assays using a recombinant human APT1 (hAPT1) and investigated the subcellular localization. For this purpose, an assay for thioesterase activity against a synthetic palmitoyl peptide using liquid chromatography/mass spectrometry was established. The thioesterase activity of hAPT1 was most active at neutral pH, and did not require Ca(2+) for its maximum activity. The K(M) values for thioesterase and lysophospholipase (against lysophosphatidylcholine) activities were 3.49 and 27.3 microM, and the V(max) values were 27.3 and 1.62 micromol/min/mg, respectively. Thus, hAPT1 revealed much higher thioesterase activity than lysophospholipase activity. One activity was competitively inhibited by another substrate in the presence of both substrates. Immunocytochemical and Western blot analyses revealed that endogenous and overexpressed hAPT1 were mainly localized in the cytosol, while some signals were detected in the plasma membrane, the nuclear membrane and ER in HEK293 cells. These results suggest that eliminating palmitoylated proteins and lysophospholipids from cytosol is one of the functions of hAPT1.

Protein purification and cloning of diacylglycerol lipase from rat brain.

Diacylglycerol (DG) lipase, which hydrolyses 1-stearoyl-2-arachidonyl-sn-glycerol to produce an endocannabinoid, 2-arachidonoylglycerol, was purified from the soluble fraction of rat brain lysates. DG lipase was purified about 1,200-fold by a sequential column chromatographic procedure. Among proteins identified by mass spectrometry analysis in the partially purified DG lipase sample, only DDHD domain containing two (DDHD2), which was formerly regarded as a phospholipase A1, exhibited significant DG lipase activity. Rat DDHD2 expressed in Chinese hamster ovary cells showed similar enzymatic properties to partially purified DG lipase from rat brain. The source of DG lipase activity in rat brain was immunoprecipitated using anti-DDHD2 antibody. Thus, we concluded that the DG lipase activity in the soluble fraction of rat brain is derived from DDHD2. DDHD2 is distributed widely in the rat brain. Immunohistochemical analysis revealed that DDHD2 is expressed in hippocampal neurons, but not in glia.

Identification and Analysis of Two Splice Variants of Human G2A Generated by Alternative Splicing

G2A is a G protein-coupled receptor that can be induced by various stressors. G2A is reported to have proton-sensing activity that mediates intracellular inositol phosphate (IP) accumulation with decreasing pH. We previously showed that G2A is also activated by some oxidized free fatty acids such as 9-hydroxyoctadecadienoic acid (9-HODE). In this study, we identified a novel alternative splice variant of G2A (G2A-b) that has a partially different N terminus compared with the G2A originally reported (G2A-a). The two splice variants of G2A show similar tissue distributions, but G2A-b is expressed more abundantly. There was no difference between the two variants in 9-HODE-induced cellular responses, such as intracellular calcium mobilization and GDP/GTP exchange of Gα protein, and in proton-sensitive IP accumulation. However, G2A-b showed a higher basal activity in terms of IP accumulation. Mutagenesis study revealed that the difference in the basal activity is attributable to the K7 residue that exists only in G2A-a. We further demonstrated that an R42A mutation largely impaired both the basal and proton-sensing activities, but did not affect the 9-HODE-induced intracellular calcium increase. Taken together, we found an additional novel G2A variant (G2A-b) that is the major transcript with functional response to ligand stimulation as well as G2A-a, and succeeded in discriminating proton-sensing and oxidized fatty acid-sensing activities of G2A.

Glucagon plays an important role in the modification of insulin secretion by leptin.

Obese people show marked hyerinsulinemia, but the exact mechanism has not been clarified. Hyperleptinemia is one of possible candidates, although there is an obvious difference in the effect of leptin on insulin secretion between isolated pancreatic islets and β-cell line. Since glucagon may modulate the effect of leptin on insulin secretion, we determined the influences of glucagon in the leptin effect on insulin secretion. The influences of glucagon in the leptin effect on insulin secretion for 10 minutes were determined by using isolated mouse islets and HIT-T 15 cells. The influences of 3-isobutyl-1- methylxanthine (IBMX), forskolin, and dibutyryl cyclic AMP were investigated in the leptin effect on insulin secretion. Leptin-inhibited insulin and glucagon secretion in isolated mouse pancreatic islets. In contrast, leptin stimulated insulin secretion in isolated mouse islets previously incubated with monoclonal anti-glucagon antibodies for 18 hours. In HIT-T 15 cells, leptin dose-dependently increased insulin secretion, but this effect was attenuated by the addition of glucagon. The stimulatory effect of leptin on insulin secretion was attenuated by 48 hour pre-incubation with glucagon. In the presence of 100 mM IBMX, leptin decreased insulin secretion from HIT-T 15 cells. Leptin also reduced insulin secretion in the presence of 1mM forskolin or 1mM dibutyryl cyclic AMP. The leptin effects on insulin secretion were affected by the existence of glucagon. Intracellular cyclic AMP concentrations may determine the leptin effects on insulin secretion in pancreatic β-cells.

Optimal cut-off value for equol-producing status in women: The Japan Nurses’ Health Study urinary isoflavone concentration survey

Equol is one of the most active soy isoflavones. When the association between soy food intake in daily life and health outcomes is examined in epidemiological studies, it is important to define the equol-producing status of each individual. However, few studies have assessed equol-producing status without a soy challenge test. To determine a robust cutoff criterion for equol producer classification in observational studies, we conducted a urinary isoflavone concentration survey in daily life among women. Furthermore, we examined the association between eating habits regarding soy foods and equol-producing status. A total of 4,412 participants were included in the analyses. Urinary isoflavones were analyzed using a high-performance liquid chromatography method. We examined the distribution of the log10 equol/daidzein ratios, finding a mixture of two normal distributions, corresponding to equol producer and non-producer subpopulations. Applying a finite mixture model, we estimated the means, standard deviations, and mixing proportions of these two distributions. The estimation was carried out using the SAS NLIN procedure. The optimal cutoff point for the log10 equol/daidzein ratio in the study population was determined to be −1.42, according to the estimated parameters of the mixture distribution. Based on this criterion, 1,830 (41.5%) of the participants were identified as equol producers. Compared with non-consumers of soy foods, consumers of soy foods had significantly higher odds of being equol producers. Using log10-transformed equol/daidzein ratios ≥ −1.42 to define equol producers among Japanese women is reasonable and suitable for determining equol-producing status in epidemiological studies. We found that soy food eating habits were associated with equol-producing status. Further investigation is required to evaluate associations between equol-producing status in daily life and health outcomes. The results of this study suggest the best cutoff point to use in the definition of equol-producing status in daily life.

Study of Butyrate Signal Transduction Pathways in Rat Hepatic Stem-Like Cells

Short-chain fatty acids (SCFAs), acetate, propionate and butyrate, are the major anions in the lumen of the large intestine. One of the G protein coupled receptors, GPR43, was recently described as a receptor for SCFAs. To understand pharmacological role of butyrate through GPR43, we analyzed cell shape, GPR43 mRNA expression and mitogen-activated protein (MAP) kinase activation in rat hepatic stem-like (HSL) cells. RT-PCR demonstrated that GPR43 mRNA was expressed in HSL cells and the expression level was not affected by butyrate treatment. Western blot analysis revealed that ERK phosphorylation was enhanced 10 min after sodium butyrate treatment then decreased to the initial level after 24 h. In contrast, Ark phosphorylation was not detected. The results obtained were contrary to our intention, because much cell death was observed after sodium butyrate treatment.