Junichi Nakagawa

Stimulation of glucose uptake in muscle cells by prolonged treatment with scriptide, a histone deacetylase inhibitor.

Glucose incorporation is regulated mainly by GLUT4 in skeletal muscles. Here we report that treatment of L6 myotubes with scriptide, a hydroxamic acid-based histone deacetylase (HDAC) inhibitor, stimulated 2-deoxyglucose uptake. The effect appeared only after 24 hr, resulting in 2.4-fold glucose uptake at treatment day 6. Scriptide acted synergistically with insulin, indicating it stimulated a distinct pathway from the insulin signaling pathway. It was not observed in undifferentiated myoblasts or 3T3-L1 adipocytes, suggesting a muscle-specific effect of scriptide. A five-carbon chain and hydroxamic acid, essential for histone deacetylase inhibition, were indispensable for this effect, and trichostatin A stimulated glucose uptake as well. Scriptide increased the cellular content of GLUT4, and induced GLUT4 translocation, but GLUT4 mRNA level did not change, indicating scriptide functions posttranslationally. Our results indicated a novel function for HDAC inhibitors of increasing GLUT4 content and its translocation in muscle cells, resulting in stimulation of glucose uptake.

Dicarbonyl/L-xylulose reductase: a potential biomarker identified by laser-capture microdissection-micro serial analysis of gene expression of human prostate adenocarcinoma.

To identify genes involved in prostate carcinogenesis, we used laser-capture microdissection-micro serial analysis of gene expression to construct libraries of paired cancer and normal cells from human tissue samples. After computational comparison of the two libraries, we identified dicarbonyl/l-xylulose reductase (DCXR), an enzyme that catalyzes α-dicarbonyl and l-xylulose, as being significantly up-regulated in prostate cancer cells. The specificity of DCXR up-regulation for prostate cancer tissues was confirmed by quantitative real-time reverse transcriptase-PCR, virtual Northern blot, and Western blot analyses. Furthermore, DCXR expression at the protein level was assessed using fresh-frozen tissues and a tissue microarray consisting of 46 cases of organ-confined early-stage prostate cancer and 29 cases of chemohormonally treated prostate cancer. In most normal prostate epithelial cells, DCXR was expressed at low levels and was localized predominantly in the cytoplasmic membrane. In contrast, in virtually all grades of early-stage prostate cancer and in all chemohormonally treated cases, DCXR was strikingly overexpressed and was localized predominantly in the cytoplasm and nucleus. In all samples, the stromal cells were completely devoid of DCXR expression. Based on these findings, we suggest that DCXR overexpression has the potential to be an additional useful biomarker for prostate cancer. (Cancer Epidemiol Biomarkers Prev 2007;16(12):2615–22)

Molecular cloning, expression and tissue distribution of hamster diacetyl reductase. Identity with L-xylulose reductase.

Using rapid amplification of cDNA ends PCR, a cDNA species for diacetyl reductase (EC 1.1.1.5) was isolated from hamster liver. The encoded protein consisted of 244 amino acids, and showed high sequence identity to mouse lung carbonyl reductase and hamster sperm P26h protein, which belong to the short-chain dehydrogenase/reductase family. The enzyme efficiently reduced L-xylulose as well as diacetyl, and slowly oxidized xylitol. The K(m) values for L-xylulose and xylitol were similar to those reported for L-xylulose reductase (EC 1.1.1.10) of guinea pig liver. The identity of diacetyl reductase with L-xylulose reductase was demonstrated by co-purification of the two enzyme activities from hamster liver and their proportional distribution in other tissues.

Suppression of AGE Precursor Formation Following Unilateral Ureteral Obstruction in Mouse Kidneys by Transgenic Expression of α-Dicarbonyl/ L -Xylulose Reductase

Unilateral ureteral obstruction (UUO) of kidneys causes acute generation of carbonyl stress. By electrospray ionization/liquid chromatography/mass spectrometry (ESI/LC/MS) we measured the content of methyl glyoxal, glyoxal, and 3-deoxyglucosone in mouse kidney extracts following UUO. UUO resulted in elevation of these dicarbonyls in the obstructed kidneys. Furthermore, the accumulation of 3-deoxyglucosone was significantly reduced in the kidneys of mice transgenic for α-dicarbonyl/L-xylulose reductase (DCXR) as compared to their wild-type littermates, demonstrating 4.91±2.04 vs. 6.45±1.85 ng/mg protein (P=0.044) for the obstructed kidneys, and 3.68±1.95 vs. 5.20±1.39 ng/mg protein (P=0.026) for the contralateral kidneys. On the other hand, collagen III content in kidneys showed no difference as monitored by in situ hybridization. Collectively, DCXR may function in the removal of renal α-dicarbonyl compounds under oxidative circumstances, but it was not sufficient to suppress acute renal fibrosis during 7 d of UUO by itself.

Suppression of Renal α-Dicarbonyl Compounds Generated following Ureteral Obstruction by Kidney-Specific α-Dicarbonyl/l-Xylulose Reductase

Renal unilateral ureteral obstruction (UUO) causes acute generation of α‐dicarbonyl stress substances, such as glyoxal, 3‐deoxyglucosone, and methylglyoxal, in the kidneys. These α‐dicarbonyl compounds are prone to form advanced glycation end products (AGEs) via the nonenzymatic Maillard reaction. Using transgenic (Tg) mice overexpressing a kidney‐specific short‐chain oxidoreductase, α‐dicarbonyl/L‐xylulose reductase (DCXR), we measured generation of α‐dicarbonyls following UUO by means of electrospray ionization/liquid chromatography/mass spectrometry in their kidney extracts. The accumulation of 3‐deoxyglucosone was significantly reduced in the kidneys of the mice Tg for DCXR compared to their wild‐type littermates, demonstrating 4.91u2003±u20032.04 vs. 6.45u2003±u20031.85u2003ng/mg protein (Pu2003=u20030.044) for the obstructed kidneys, and 3.68u2003±u20031.95 vs. 5.20u2003±u20031.39u2003ng/mg protein (Pu2003=u20030.026) for the contralateral kidneys. Despite the reduction in accumulated α‐dicarbonyls, collagen III content in kidneys of the Tg mice and their wild‐type littermates showed no difference as monitored by in situ hybridization. Collectively, DCXR may function in the removal of renal α‐dicarbonyl compounds under oxidative circumstances, but it is not sufficient to suppress acute renal fibrosis during 7u2003days UUO.

Identification of amino acid residues involved in substrate recognition of L-xylulose reductase by site-directed mutagenesis.

L-Xylulose reductase (XR) catalyzes the oxidoreduction between xylitol and L-xylulose in the uronate cycle. The enzyme has been shown to be identical to diacetyl reductase, an enzyme that reduces alpha-dicarbonyl compounds. XR belongs to the short-chain dehydrogenase/reductase family, and shows high sequence identity with mouse lung carbonyl reductase (MLCR), an enzyme that reduces 3-ketosteroids but not sugars. In this study, we have confirmed the roles of Ser136, Tyr149 and Lys153 of XR as the catalytic triad by drastic loss of activity resulting from the mutagenesis of S136A, Y149F and K153M in rat XR. We have also constructed several mutant XRs, in which putative substrate binding residues from rat XR were substituted with those found in the corresponding positions of MLCR, in order to identify amino acids responsible for the different substrate recognition of the enzymes. While single mutants at positions 137, 143, 146, 190 and 191 caused little or moderate change in substrate specificity, a double mutant (N190V and W191S) and triple mutant (Q137M, L143F and H146L) resulted in almost loss of activity for only the sugars. In addition, the triple mutant exhibited 3-ketosteroid reductase activity, which was further enhanced by quintuple mutagenesis of the above five residues. These results suggest the importance of the size and hydrophobicity of the five residues for substrate recognition by XR and MLCR. Furthermore, the mutant enzymes containing a Q137M mutation were stable against cooling, which provides a structural mechanism of the cold inactivation that is a characteristic of the rodent XR.

Crystallization and preliminary crystallographic analysis of human l-xylulose reductase

Human L-xylulose reductase was crystallized from buffered polyethylene glycol solutions using the hanging-drop vapour-diffusion method. The crystals diffract to 2.1 A resolution and belong to the orthorhombic P222 space group, with unit-cell parameters a = 72.9, b = 74.1, c = 87.9 A. This is the first crystallization report of a xylulose reductase that is identical to diacetyl reductase.