Effects of ethyl hexanoate on activities of sympathetic nerves innervating the brown and white adipose tissues, body temperature, and plasma fatty acids.

The autonomic nervous system (ANS) is implicated in maintaining homeostasis of the internal environment in mammals. Therefore, changes occurring in the ANS can cause alterations of physiological phenomena. Ethyl hexanoate (EH) is known as the aroma component of apples. To study the action of ethyl hexanoate on physiological phenomena, we examined the effect of an intragastric (IG) injection of 1 mL/kg body weight of 0.1 ppm EH solution on sympathetic nerve activity innervating the brown adipose tissue (BAT) and white adipose tissue (WAT) in anesthetized rats. Consequently, IG administration of EH increased activity of the sympathetic nerves innervating both the BAT and WAT. In addition, the effects of the IG injection on body temperature above the interscapular BAT and plasma free fatty acid (FFA) concentration were also examined in conscious rats. In this attempt IG injection of EH elevated both the body temperature and plasma FFA levels. Furthermore, subdiaphragmatic vagotomy eliminated the effects of EH on sympathetic nerves innervating BAT and WAT. These findings suggest that EH causes excitations of sympathetic nerves innervating BAT and WAT, and enhances thermogenesis and lipolysis via the afferent vagus nerve. Thus, these present findings also suggest the possibility that EH might have anti-obesity effects.

Topical application of L-carnosine to skeletal muscle excites the sympathetic nerve innervating the contralateral skeletal muscle in rats.

We previously obtained evidence suggesting that physical exercise increases the release of L-carnosine (CAR) from muscles and that CAR affects autonomic neurotransmission and physiological phenomena in rats. It has also been reported that exercise elicits an increase in activity of the sympathetic nerve innervating the skeletal muscle. Therefore, in this study, we investigated the effect of CAR application, onto the surface of the right femoral muscle, on activity of the sympathetic nerve innervating the left femoral muscle, in urethane-anesthetized rats. Topical application of 10 pg (44.2 fmol) of CAR increased either skeletal muscle sympathetic nerve activity (skeletal muscle-SNA) or skeletal muscle blood flow (skeletal muscle-BF) of the contralateral skeletal muscle. Furthermore, thioperamide, a histamine H3-antagonist, inhibited the increase in skeletal muscle-SNA, and butoxamine, a ß2-antagonist, abolished the increase in skeletal muscle-BF caused by topical application of CAR. The present results suggest that CAR released from muscles during physical exercise might affect skeletal muscle-SNA and skeletal muscle-BF on the opposite side of the body via a CAR evoked effect in muscles.

Aldehyde dehydrogenase, Ald4p, is a major component of mitochondrial fluorescent inclusion bodies in the yeast Saccharomyces cerevisiae.

When Saccharomyces cerevisiae strain 3626 was cultured to the stationary phase in a medium that contained glucose, needle-like structures that emitted autofluorescence were observed in almost all cells by fluorescence microscopy under UV excitation. The needle-like structures completely overlapped with the profile of straight elongated mitochondria. Therefore, these structures were designated as mitochondrial fluorescent inclusion bodies (MFIBs). The MFIB-enriched mitochondrial fractions were successfully isolated and 2D-gel electrophoresis revealed that a protein of 54 kDa was only highly concentrated in the fractions. Determination of the N-terminal amino acid sequence of the 54-kDa protein identified it as a mitochondrial aldehyde dehydrogenase, Ald4p. Immunofluorescence microscopy showed that anti-Ald4p antibody specifically stained MFIBs. Freeze-substitution electron microscopy demonstrated that cells that retained MFIBs had electron-dense filamentous structures with a diameter of 10 nm in straight elongated mitochondria. Immunoelectron microscopy showed that Ald4p was localized to the electron-dense filamentous structures in mitochondria. These results together showed that a major component of MFIBs is Ald4p. In addition, we demonstrate that MFIBs are common features that appear in mitochondria of many species of yeast.

Involvement of ST6Gal I in the biosynthesis of a unique human colon cancer biomarker candidate, alpha2,6-sialylated blood group type 2H (ST2H) antigen.

The alpha2,6-sialylated blood group type 2H (ST2H) antigen (Fucalpha1-2(NeuAcalpha2-6)Galbeta1-4GlcNAcbeta1-3Galbeta1-4Glc-Cer) is a fucoganglioside found in human colon cancer tissues. To elucidate an enzyme responsible for the ST2H antigen formation, we screened some partially purified candidate enzymes, alpha2,6-sialyltransferases, ST6Gal I and ST6Gal II, and alpha1,2-fucosyltransferases, FUT1 and FUT2 for their activities towards pyridylaminated type 2H (Fucalpha1-2Galbeta1-4GlcNAcbeta1-3Galbeta1-4Glc-PA) or LS-tetrasaccharide c (LST-c: NeuAcalpha2-6Galbeta1-4GlcNAcbeta1-3Galbeta1-4Glc-PA) as acceptor substrates. Here we show the ST6Gal I transfers NeuAc from the donor CMP-NeuAc to the terminal Gal of PA-type 2H, which formed the ST2H antigen, but the others could not synthesize it. Using a recombinant ST6Gal I, enzymatic reactions with two types of acceptors, PA-type 2H and PA-lacto-N-neotetraose (LNnT), were kinetically analysed. On the basis of catalytic efficiency (V(max)/K(m)), the specificity of ST6Gal I towards the PA-type 2H was estimated to be 42 times lower than that for PA-LNnT. The overexpression of ST6Gal I in human colon cancer DLD-1 cells effectively resulted in the ST2H antigen formation, as judged by LC-ESI-IT-MS. Many lines of evidence suggest the up-regulation of ST6Gal I in human colon cancer specimens. Collectively, these findings indicate that ST6Gal I is responsible for ST2H antigen biosynthesis in human colon cancer cells.

Novel ganglioside found in adenocarcinoma cells of Lewis-negative patients.

We have precisely analyzed the structures of glycosphingolipids of human cancer cells and normal epithelial cells using several methods, including enzymatic release of carbohydrate moieties, fluorescent labeling, and identification using 2D mapping, enzymatic digestion, and mass spectrometry. These analyses enabled the identification of novel tumor-associated carbohydrate antigens that can be used to elucidate the involvement of carbohydrates in cancer malignancy and could act as candidate tumor markers. In our previous study, we identified a novel glycosphingolipid that accumulates in colon cancer cells, NeuAcα2-6(Fucα1-2)Galß1-4GlcNAcß1-3Galß1-4Glc (α2-6 sialylated type 2H, ST2H). Here, structural analyses of cancer cells and normal epithelial cells from 60 colorectal and five pancreatic cancer patients, including four and two Lewis-negative individuals, respectively, reveal the presence of an additional novel glycosphingolipid, NeuAcα2-6(Fucα1-2)Galß1-3GlcNAcß1-3Galß1-4Glc (α2-6 sialylated type 1H, ST1H). ST2H was found in colorectal and pancreatic cancer cells from about half of the cases. Unlike ST2H, ST1H was found in cancer cells from three out of six Lewis-negative patients (i.e., two cases of colorectal and one case of pancreatic cancer). However, the moiety was not found in normal epithelial cells or cancer cells from 59 Lewis-positive patients. These findings suggest that the accumulation of this carbohydrate antigen occurs predominantly in cancer cells of Lewis-negative patients. When the ST1H epitope is also carried on mucins as well as glycosphingolipids, this epitope is a promising tumor marker candidate, especially for Lewis-negative individuals.

Unusual accumulation of sulfated glycosphingolipids in colon cancer cells.

The structures of glycosphingolipids from highly purified colorectal cancer cells and normal colorectal epithelial cells of 16 patients have been analyzed in fine detail (Misonou Y, Shida K, Korekane H, Seki Y, Noura S, Ohue M, Miyamoto Y. 2009. Comprehensive Clinico-Glycomic Study of 16 Colorectal Cancer Specimens: Elucidation of aberrant glycosylation and ts mechanistic causes in colorectal cancer cells. J Proteome Res. 8:2990-3005). Further structural analyses demonstrated that colon cancer cells from two patients accumulated unusual glycosphingolipids which were not observed in either colorectal cancer cells or normal colorectal epithelial cells from the other patients. Mass spectrometry analyses revealed that the unusual structures include sulfated oligosaccharides. The structures of the glycosphingolipids of the cancer cells from these two cases were analyzed by methods which include enzymatic release of carbohydrate moieties, fluorescent labeling with aminopyridine and identification using two-dimensional mapping, enzymatic digestion and mass spectrometry together with methanolysis, and the use of newly synthesized sulfo-fucosylated oligosaccharides as standards. The colon cancer cells from one of the patients demonstrate a variety of oligosaccharides as major components which are sulfated at the C6 position of subterminal GlcNAc and at C3 positions of terminal galactose with or without sialylation or fucosylation. These include 6-sulfo Le(x), 6'-sialyl 6-sulfo lactosamine, and 3'-sialyl 6-sulfo Le(x), in addition to sialylated or fucosylated derivatives of type-1 and type-2 hybrid oligosaccharides. The colon cancer cells from the other patient have two kinds of sulfated oligosaccharides, a 6-sulfo Le(x) structure and a 3'-sulfo Le(x) structure, as minor components. Taking into consideration the clinical features of the two patients, the biological significance of sulfated glycosphingolipids on cancer cells is discussed.

Comprehensive clinico-glycomic study of 16 colorectal cancer specimens: elucidation of aberrant glycosylation and its mechanistic causes in colorectal cancer cells.

The structures of neutral and acidic glycosphingolipids from both normal colorectal epithelial cells and colorectal cancer cells, which were highly purified with the epithelial cell marker CD326, have been analyzed. The analysis was performed on samples from 16 patients. The carbohydrate moieties from glycosphingolipids were released by endoglycoceramidase II, labeled by pyridylamination, and identified using two-dimensional mapping and mass spectrometry. The structures from normal colorectal epithelial cells are characterized by dominant expression of neutral type-1 chain oligosaccharides. Three specific alterations were observed in malignant transformation; increased ratios of type-2 oligosaccharides, increased alpha2-3 and/or alpha2-6 sialylation and increased alpha1-2 fucosylation. Although the degree of alteration varies case to case, we found that two characteristic alterations tend to be associated with clinical features. One is a shift from type-1 dominant normal colorectal epithelial cells to type-2 dominant colorectal cancer cells. This shift was found in 5 patients having hepatic metastasis. The other is specific elevation of alpha2-3 sialylation observed in 2 cases exhibiting high serum levels of CA19-9. Examination of the activities of the related glycosyltransferases revealed that while some alterations could be accounted for by changes in the activities of related glycosyltransferases others could not. Although the number of cases analyzed is small, these findings provide valuable information which will help in the elucidation of the mechanism of synthesis of aberrant glycosylation and its involvement in cancer malignancy.

Acrolein induces cyclooxygenase-2 and prostaglandin production in human umbilical vein endothelial cells: roles of p38 MAP kinase.

OBJECTIVE: Acrolein, a known toxin in tobacco smoke, might be involved in atherogenesis. This study examined the effect of acrolein on expression of cyclooxygenase-2 (COX-2) and prostaglandin (PG) production in endothelial cells. METHODS AND RESULTS: Cyclooxygenase (COX)-2 induction by acrolein and signal pathways were measured using Western blots, Northern blots, immunofluorescence, ELISA, gene silencing, and promoter assay. Colocalization of COX2 and acrolein-adduct was determined by immunohistochemistry. Here we report that the levels of COX-2 mRNA and protein are increased in human umbilical vein endothelial cells (HUVECs) after acrolein exposure. COX-2 was found to colocalize with acrolein-lysine adducts in human atherosclerotic lesions. Inhibition of p38 MAPK activity abolished the induction of COX-2 protein and PGE2 accumulation by acrolein, while suppression of extracellular signal-regulated kinase (ERK) and JNK activity had no effect on the induction of COX-2 expression in experiments using inhibitors and siRNA. Furthermore, rottlerin, an inhibitor of protein kinase Cdelta (PKCdelta), abrogated the upregulation of COX-2 at both protein and mRNA levels. CONCLUSION: These results provide that acrolein may play a role in progression of atherosclerosis and new information on the signaling pathways involved in COX-2 upregulation in response to acrolein and provide evidence that PKCdelta and p38 MAPK are required for transcriptional activation of COX-2.

The internalization and metabolism of 3-deoxyglucosone in human umbilical vein endothelial cells.

3-Deoxyglucosone (3-DG), a dicarbonyl compound produced by glycation, plays a role in the modification and cross-linking of long-lived proteins. We synthesized [3H]3-DG from [3H]glucose and developed an internalization assay system using HPLC to examine its cellular metabolism. When smooth muscle cells or human umbilical vein endothelial cells were incubated with [3H]3-DG, it was found that [3H]3-DG was internalized by cells in a time dependent manner. The rate of internalization was reduced when the cells were incubated at 4 degrees C or treated with phenylarsine oxide (PAO). By monitoring [3H]3-DG taken up by cells, it was confirmed that 3-DG is reduced to 3-deoxyfructose (3-DF) and that this reaction was inhibited by an aldo-keto reductase inhibitor (ARI). The presence of 3-DG led to an increase in reactive oxygen species levels in the cells and subsequent apoptosis, and the effect was enhanced by pretreatment with ARI. These results suggest that 3-DG is internalized by cells and reduced to 3-DF by aldo-keto reductases, and that the internalized 3-DG is responsible for the production of intracellular oxidative stress.

Acrolein produces nitric oxide through the elevation of intracellular calcium levels to induce apoptosis in human umbilical vein endothelial cells: implications for smoke angiopathy.

Acrolein is a highly electrophilic alpha, beta-unsaturated aldehyde, the levels of which are increased in the blood of smokers. To determine if acrolein is involved in the pathology of smoke angiopathy, the effect of acrolein on human umbilical vein endothelial cells (HUVEC) was examined. Intracellular nitric oxide (NO) levels, determined using diaminofluorescein-2 diacetate (DAF-2 DA), an NO sensitive fluorescent dye, were found to be increased after treatment in HUVEC with 10 microM acrolein. The measurement of nitrite with 2,3-diaminonaphthalene and a Western blot analysis revealed that nitrite and S-nitroso-cysteine levels were increased in a dose-dependent manner, confirming that NO production is increased by acrolein. The increase was not reduced by treatment with 10mM N-acetyl-l-cysteine (NAC), an anti-oxidant, but was reduced with 10 microM of the intracellular calcium chelator, 1,2-bis (o-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid tetra (acetoxymethyl) ester. Acrolein-stimulated NO production was significantly reduced by pretreatment with 1mM N(G)-nitro-l-arginine-methyl ester (L-NAME), an NO synthase inhibitor. The cytotoxicity of acrolein was reduced by pretreatment with 10 microM 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (carboxy-PTIO), an intracellular NO scavenger, or 1mM L-NAME, whereas it was not reduced by 10mM NAC, 20 microM Curcumin, another peroxide scavenger, or 100 microM Mn(III)TMPyP, a superoxide dismutase mimic. Nuclear staining and a Western blot analysis using an anti-cleaved caspase 3 antibody revealed that the reduced viability of HUVEC by acrolein was due to apoptosis, which was reversed after pretreatment with 0.1mM carboxy-PTIO or 1mM L-NAME. Thus, acrolein increases intracellular calcium production to induce intracellular NO production by a calcium-dependent NO synthase, possibly eNOS, and the excess and rapid increase in NO might lead to the apoptosis of HUVEC. These data suggest that acrolein might be involved in the pathology of smoke angiopathy through the NO-induced apoptosis of endothelial cells.

A common pathway for intracellular reactive oxygen species production by glycoxidative and nitroxidative stress in vascular endothelial cells and smooth muscle cells.

A large body of evidence suggests that carbonyl compounds induce intracellular signaling by increasing oxidative stress in the cell; however, the mechanisms involved have not been fully described. The focus of our research is on the pathway in which antioxidative enzymes are modified and inactivated by carbonyl compounds, resulting in the accumulation of active oxygen species in the cell. A common pathway appears to exist for cellular signaling evoked by nitroxidative stress. It could be concluded that some glycoxidative stress and nitroxidative stress cause intracellular signaling via similar mechanisms. The elucidation of the pathway for extracellular stress-induced reactive oxygen species (ROS) production would be important for our understanding of the role of ROS as signaling molecules.

Acrolein induces Hsp72 via both PKCdelta/JNK and calcium signaling pathways in human umbilical vein endothelial cells.

Acrolein is a highly electrophilic alpha,beta-unsaturated aldehydes to which humans are exposed in a variety of environment situations and is also a product of lipid peroxidation. Increased levels of unsaturated aldehydes play an important role in the pathogenesis of a number of human diseases such as Alzheimer's disease, atherosclerosis and diabetes. A number of studies have reported that acrolein evokes downstream signaling via an elevation in cellular oxidative stress. Here, we report that low concentrations of acrolein induce Hsp72 in human umbilical vein endothelial cells (HUVEC) and that both the PKCdelta/JNK pathway and calcium pathway were involved in the induction. The findings confirm that the production of reactive oxygen species (ROS) is not directly involved in the pathway. The induction of Hsp72 was not observed in other cells such as smooth muscle cells (SMC) or COS-1 cells. The results suggest that HUVEC have a unique defense system against cell damage by acrolein in which Hsp72 is induced via activation of both the PKCd/JNK and the calcium pathway.

Induction of thioredoxin reductase as an adaptive response to acrolein in human umbilical vein endothelial cells.

Acrolein is a highly electrophilic alpha,beta-unsaturated aldehyde to which humans are exposed in a variety of environment situations and is also a product of lipid peroxidation. Increased unsaturated aldehyde levels and reduced antioxidant status play an important role in the pathogenesis of a number of human diseases such as Alzheimer's, atherosclerosis, and diabetes. Mammalian thioredoxin reductase (TR), a central antioxidant enzyme, is a selenoprotein that catalyzes the reduction of oxidized thioredoxin. The findings reported here show that low concentrations of acrolein rapidly inactivate TR, both in vitro and in vivo. These data suggest that acrolein may directly inactivate TR, resulting in an increase in oxidative cellular damage. In addition, we also found that the initial inactivation of TR molecules by acrolein triggers a compensatory signal for inducing TR gene expression in human umbilical vein endothelial cells (HUVEC). The results of the present study suggest that HUVEC may have a protective system against cell damage by acrolein via the upregulation of TR, which is an adaptive response to oxidative stress.

Oxidative stress caused by inactivation of glutathione peroxidase and adaptive responses.

Reactive oxygen species (ROS) are generated as by-products of cellular metabolism, primarily in the mitochondria. When the cellular production of ROS exceeds the cell's antioxidant capacity, cellular macromolecules such as lipids, proteins and DNA can be damaged. Because of this, 'oxidative stress' is thought to contribute to aging and pathogenesis of a variety of human diseases. However, in the last 10-15 years, a considerable body of evidence has accumulated that ROS serve as subcellular messengers, and play a role in gene regulation and signal transduction pathways, which may be involved in defensive mechanisms against oxidative stress. This review focuses on oxidative stress caused by the inactivation of glutathione peroxidase (GPx), a major peroxide scavenging enzyme. GPx is inactivated by a variety of physiological substances, including nitric oxide and carbonyl compounds in vitro and in cell culture. Decreased GPx activity has also been reported in tissues where oxidative stress occurs in several pathological animal models. The accumulation of increased levels of peroxide resulting from inactivation of GPx may act as a second messenger and regulate expression of anti-apoptotic genes and the GPx itself to protect against cell damage. These findings suggest that GPx undergoes inactivation under various conditions such as nitroxidative stress and glycoxidative stress, and that these changes are a common feature of various types of oxidative stress which may be associated with the modification of redox regulation and cellular function.

Glycation proceeds faster in mutated Cu, Zn-superoxide dismutases related to familial amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) involves the progressive degeneration of motor neurons in the spinal cord and motor cortex. It has been shown that 15-20% of patients with familial ALS (FALS) have defects in the Sod1 gene that encodes Cu, Zn-superoxide dismutase (SOD). To elucidate the pathological role of mutated Cu, Zn-SODs in FALS, the susceptibility of mutants to glycation was examined. Mutated Cu, Zn-SODs (G37R, G93A, and I113T) related to FALS and wild type were produced in a baculovirus/insect cell expression system. Glycated and nonglycated proteins were separated on a boronate column, and the nonglycated fraction was then incubated with glucose. The mutated Cu, Zn-SODs were found to be highly susceptible to glycation compared with the wild-type enzyme as estimated by Western blot analysis using an anti-hexitol lysine antibody. The mutated Cu, Zn-SOD incubated with glucose generated higher levels of hydrogen peroxide than the wild-type enzyme. Mutated Cu, Zn-SODs were also shown to be highly susceptible to fructation, and the fructated mutant also produced higher levels of hydrogen peroxide than the wild type. These results suggest that high susceptibility of mutated Cu, Zn-SODs to glycation could be the origin of the oxidative stress associated with neuronal dysfunction in FALS.