Ikuyo Ichi

S1P3-mediated cardiac fibrosis in sphingosine kinase 1 transgenic mice involves reactive oxygen species

AIMS Sphingosine kinase 1 (SPHK1), its product sphingosine-1-phosphate (S1P), and S1P receptor subtypes have been suggested to play protective roles for cardiomyocytes in animal models of ischaemic preconditioning and cardiac ischaemia/reperfusion injury. To get more insight into roles for SPHK1 in vivo, we have generated SPHK1-transgenic (TG) mice and analysed the cardiac phenotype. METHODS AND RESULTS SPHK1-TG mice overexpressed SPHK1 in diverse tissues, with a nearly 20-fold increase in enzymatic activity. The TG mice grew normally with normal blood chemistry, cell counts, heart rate, and blood pressure. Unexpectedly, TG mice with high but not low expression levels of SPHK1 developed progressive myocardial degeneration and fibrosis, with upregulation of embryonic genes, elevated RhoA and Rac1 activity, stimulation of Smad3 phosphorylation, and increased levels of oxidative stress markers. Treatment of juvenile TG mice with pitavastatin, an established inhibitor of the Rho family G proteins, or deletion of S1P3, a major myocardial S1P receptor subtype that couples to Rho GTPases and transactivates Smad signalling, both inhibited cardiac fibrosis with concomitant inhibition of SPHK1-dependent Smad-3 phosphorylation. In addition, the anti-oxidant N-2-mercaptopropyonylglycine, which reduces reactive oxygen species (ROS), also inhibited cardiac fibrosis. In in vivo ischaemia/reperfusion injury, the size of myocardial infarct was 30% decreased in SPHK1-TG mice compared with wild-type mice. CONCLUSION These results suggest that chronic activation of SPHK1-S1P signalling results in both pathological cardiac remodelling through ROS mediated by S1P3 and favourable cardioprotective effects.

Association of ceramides in human plasma with risk factors of atherosclerosis

Atherosclerosis is a multifactorial disorder. Recent studies indicate that the plasma level of sphingomyelin, which yields ceramide, correlates with the risk of coronary heart disease. Therefore, ceramide, a well-known lipid causing apoptosis in various cell types, may contribute to atherogenesis. We examined the relationship between ceramide concentration and risk factors of atherosclerosis in normal human plasma using electrospray tandem mass spectrometry (LC-MS/MS). Major ceramides in human plasma were C24∶0 and C24∶1. The ceramide concentration showed a significant positive correlation with total cholesterol (TC) and triglycerides (TG). In addition, plasma ceramide level increased drastically at a high level of LDL cholesterol (more than 170 mg/dL). Our previous studies demonstrated that the sum of fragmented and conjugated apolipoprotein B-100 proteins (B-ox), which were products of a radical reaction of LDL as well as plasma, was a reliable index of atherosclerosis. B-ox showed a significant positive correlation with the plasma ceramide level. Based on these results, we propose that the ceramide level in human plasma is a risk factor at the early stages of atherosclerosis.

Neutral sphingomyelinase-induced ceramide accumulation by oxidative stress during carbon tetrachloride intoxication

Ceramide is a biologically active lipid causing apoptosis in a variety of cells. In this study, we examined the effect of CCl4 on the ceramide metabolism and indicators of oxidative stress. After 12 h of oral administration of CCl4 (4 ml/kg body weight as a 1:1 mixture of CCl4 and mineral oil) to rats, aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were increased. Antioxidants such as vitamins C and E were decreased in the liver and kidney. In addition, the ratio of GSH/GSSG in the liver, plasma, kidney, and brain decreased at 2h. The total ceramide in the liver significantly increased as early as 2h after CCl4 administration. After 24 and 36 h, the total ceramide in plasma and the kidney was also augmented. In the brain, the total ceramide dramatically increased at 36 h. These results suggested that the increased ceramide in plasma was transferred to the kidney and the brain. The activity of neutral sphingomyelinase (SMase), which was reported to be enhanced by the decrease of GSH, was significantly increased after CCl4 treatment in the liver, kidney, and brain. However, acid SMase activities were not increased in the liver and kidney. Thus, the activation of neutral SMase via oxidative stress induced the increase of ceramide during CCl4 intoxication in not only the liver but also other tissues. These results suggested that the excess accumulation of ceramide causes damage in other organs including the kidney and brain during fulminant hepatic failure.

Perilipin-Mediated Lipid Droplet Formation in Adipocytes Promotes Sterol Regulatory Element-Binding Protein-1 Processing and Triacylglyceride Accumulation

Sterol regulatory element-binding protein-1 (SREBP-1) has been thought to be a critical factor that assists adipogenesis. During adipogenesis SREBP-1 stimulates lipogenic gene expression, and peroxisome proliferator-activated receptor γ (PPARγ) enhances perilipin (plin) gene expression, resulting in generating lipid droplets (LDs) to store triacylglycerol (TAG) in adipocytes. Plin coats adipocyte LDs and protects them from lipolysis. Here we show in white adipose tissue (WAT) of plin−/− mice that nuclear active SREBP-1 and its target gene expression, but not nuclear SREBP-2, significantly decreased on attenuated LD formation. When plin−/− mouse embryonic fibroblasts (MEFs) differentiated into adipocytes, attenuated LDs were formed and nuclear SREBP-1 decreased, but enforced plin expression restored them to their original state. Since LDs are largely derived from the endoplasmic reticulum (ER), alterations in the ER cholesterol content were investigated during adipogenesis of 3T3-L1 cells. The ER cholesterol greatly reduced in differentiated adipocytes. The ER cholesterol level in plin−/− WAT was significantly higher than that of wild-type mice, suggesting that increased LD formation caused a change in ER environment along with a decrease in cholesterol. When GFP-SREBP-1 fusion proteins were exogenously expressed in 3T3-L1 cells, a mutant protein lacking the S1P cleavage site was poorly processed during adipogenesis, providing evidence of the increased canonical pathway for SREBP processing in which SREBP-1 is activated by two cleavage enzymes in the Golgi. Therefore, LD biogenesis may create the ER microenvironment favorable for SREBP-1 activation. We describe the novel interplay between LD formation and SREBP-1 activation through a positive feedback loop.

Identification of genes and pathways involved in the synthesis of Mead acid (20:3n − 9), an indicator of essential fatty acid deficiency

In mammals, 5,8,11-eicosatrienoic acid (Mead acid, 20:3n-9) is synthesized from oleic acid during a state of essential fatty acid deficiency (EFAD). Mead acid is thought to be produced by the same enzymes that synthesize arachidonic acid and eicosapentaenoic acid, but the genes and the pathways involved in the conversion of oleic acid to Mead acid have not been fully elucidated. The levels of polyunsaturated fatty acids in cultured cells are generally very low compared to those in mammalian tissues. In this study, we found that cultured cells, such as NIH3T3 and Hepa1-6 cells, have significant levels of Mead acid, indicating that cells in culture are in an EFAD state under normal culture conditions. We then examined the effect of siRNA-mediated knockdown of fatty acid desaturases and elongases on the level of Mead acid, and found that knockdown of Elovl5, Fads1, or Fads2 decreased the level of Mead acid. This and the measured levels of possible intermediate products for the synthesis of Mead acid such as 18:2n-9, 20:1n-9 and 20:2n-9 in the knocked down cells indicate two pathways for the synthesis of Mead acid: pathway 1) 18:1n-9→(Fads2)→18:2n-9→(Elovl5)→20:2n-9→(Fads1)→20:3n-9 and pathway 2) 18:1n-9→(Elovl5)→20:1n-9→(Fads2)→20:2n-9→(Fads1)→20:3n-9.

Regulation of Peroxisomal Lipid Metabolism by Catalytic Activity of Tumor Suppressor H-rev107

Background: Physiological function of the tumor suppressor H-rev107, showing a phospholipase A1/2 activity, is poorly understood. Results: Overexpression of the catalytically active H-rev107 in mammalian cells decreased endogenous levels of ether-type lipids and altered intracellular localization of peroxisomal markers. Conclusion: H-rev107 may enzymatically regulate peroxisomal function. Significance: These results suggest a physiological role of H-rev107 discovered as a tumor suppressor. H-rev107 is a mammalian protein belonging to the HRAS-like suppressor family. Although the protein was originally found as a tumor suppressor, currently it is receiving considerable attention as a regulator of adipocyte lipolysis. We recently revealed that purified recombinant H-rev107 has phospholipase A1/2 activity, releasing free fatty acids from glycerophospholipids with a preference for esterolysis at the sn-1 position. In the present study, we constitutively expressed H-rev107 in cloned HEK293 cells to examine its biological function in living cells. Initially, the cells accumulated free fatty acids. We also found a remarkable decrease in the levels of ether-type lipids, including plasmalogen and ether-type triglyceride, with a concomitant increase in fatty alcohols, substrates for the biosynthesis of ether-type lipids. Considering that peroxisomes are involved in the ether-type lipid biosynthesis, we next focused on peroxisomes and found that the peroxisomal markers 70-kDa peroxisomal membrane protein and catalase were abnormally distributed in the transfected cells. These biochemical and morphological abnormalities were not seen in HEK293 cells stably expressing a catalytically inactive mutant of H-rev107. When H-rev107 or its fusion protein with enhanced green fluorescence protein was transiently expressed in mammalian cells, both proteins were associated with peroxisomes in some of the observed cells. These results suggest that H-rev107 interferes with the biosynthesis of ether-type lipids and is responsible for the dysfunction of peroxisomes in H-rev107-expressing cells.

Oligo-astheno-teratozoospermia in mice lacking ORP4, a sterol-binding protein in the OSBP-related protein family.

Oligo‐astheno‐teratozoospermia (OAT), a condition that includes low sperm number, low sperm motility and abnormal sperm morphology, is the commonest cause of male infertility. Because genetic analysis is frequently impeded by the infertility phenotype, the genetic basis of many of OAT conditions has been hard to verify. Here, we show that deficiency of ORP4, a sterol‐binding protein in the oxysterol‐binding protein (OSBP)‐related protein family, causes male infertility due to severe OAT in mice. In ORP4‐deficient mice, spermatogonia proliferation and subsequent meiosis occurred normally, but the morphology of elongating and elongated spermatids was severely distorted, with round‐shaped head, curled back head or symplast. Spermatozoa derived from ORP4‐deficient mice had little or no motility and no fertilizing ability in vitro. In ORP4‐deficient testis, postmeiotic spermatids underwent extensive apoptosis, leading to a severely reduced number of spermatozoa. At the ultrastructural level, nascent acrosomes appeared to normally develop in round spermatids, but acrosomes were detached from the nucleus in elongating spermatids. These results suggest that ORP4 is essential for the postmeiotic differentiation of germ cells.

The Beneficial Effect of Propolis on Fat Accumulation and Lipid Metabolism in Rats Fed a High‐Fat Diet

This study examined whether propolis, which had many biological activities, affected body fat and lipid metabolism. Four-week-old Wistar rats were fed a control or propolis diet for 8 wk. The control group was fed a high-fat diet, the low and the high group were fed a high-fat diet supplemented with 0.5% (w/w) and 0.05% (w/w) propolis, respectively. The weight of total white adipose tissue of the high group was lower than that of the control group. The level of PPARgamma protein in the adipose tissues of the high group was significantly lower than that of the control group. In plasma and the liver, the high group showed a significantly reduced level of cholesterol and triglyceride compared to the control group. The liver PPARalpha protein level of the high group was significantly higher than that of the control group. The liver HMG-CoA reductase protein in the high group was also significantly lower than that in the control group. Results from rats on an olive oil loading test were used to investigate whether propolis inhibited triglyceride absorption. The serum triglyceride level of the group, which received propolis corresponding to the daily dose of the high group, was significantly lower than that of the control group. It is possible that the administration of propolis improves the accumulation of body fat and dyslipidemia via the change of the expression of proteins involved in adipose depot and lipid metabolism.

Changes in the phospholipid fatty acid composition of the lipid droplet during the differentiation of 3T3-L1 adipocytes.

Lipid droplets (LDs) are independent organelles in adipocytes that are composed of a lipid ester core surrounded by a phospholipid monolayer. The fatty acid composition of the phospholipid monolayer should determine the metabolism and dynamics of LDs. In this study, we examined the fatty acid composition of phospholipid monolayer in LDs during the differentiation of 3T3-L1 adipocytes. The levels of saturated fatty acids (SFAs), such as 16:0 and 18:0, in phosphatidylcholine (PC) and phosphatidylethanolamine (PE) of LDs decreased during differentiation. In contrast, the levels of monounsaturated fatty acids (MUFAs) such as 16:1n-7 and 18:1n-9 in PC and PE of LDs and the level of polyunsaturated fatty acids (PUFAs) such as 20:3n-6, 20:4n-6 and 22:6n-3 in PE of LDs increased during differentiation. These results suggest that the phospholipid monolayer in mature LDs is more fluid than that in nascent LDs. The fatty acid compositions of the LD monolayer were different from those of the microsome bilayer in the early stage of differentiation, but similar to those of the microsome bilayer in the late stage of differentiation. These data provide evidence that biophysical properties of the phospholipid monolayer in LDs change during adipocyte differentiation.

Ezetimibe in combination with statins ameliorates endothelial dysfunction in coronary arteries after stenting: the CuVIC trial (effect of cholesterol absorption inhibitor usage on target vessel dysfunction after coronary stenting), a multicenter randomized controlled trial

Objectives— We sought to investigate whether treatment with ezetimibe in combination with statins improves coronary endothelial function in target vessels in coronary artery disease patients after coronary stenting. Approach and Results— We conducted a multicenter, prospective, randomized, open-label, blinded-end point trial among 11 cardiovascular treatment centers. From 2011 to 2013, 260 coronary artery disease patients who underwent coronary stenting were randomly allocated to 2 arms (statin monotherapy, S versus ezetimibe [10 mg/d]+statin combinational therapy, E+S). We defined target vessel dysfunction as the primary composite outcome, which comprised target vessel failure during treatment and at the 6- to 8-month follow-up coronary angiography and coronary endothelial dysfunction determined via intracoronary acetylcholine testing performed in cases without target vessel failure at the follow-up coronary angiography. Coadministration of ezetimibe with statins further lowered low-density lipoprotein cholesterol levels (83±23 mg/dL in S versus 67±23 mg/dL in E+S; P<0.0001), with significant decreases in oxidized low-density lipoprotein and oxysterol levels. Among patients without target vessel failure, 46 out of 89 patients (52%) in the S arm and 34 out of 96 patients (35%) in the E+S arm were found to have coronary endothelial dysfunction (P=0.0256), and the incidence of target vessel dysfunction at follow-up was significantly decreased in the E+S arm (69/112 (62%) in S versus 47/109 (43%) in E+S; P=0.0059). A post hoc analysis of post-treatment low-density lipoprotein cholesterol–matched subgroups revealed that the incidence of both target vessel dysfunction and coronary endothelial dysfunction significantly decreased in the E+S arm, with significant reductions in oxysterol levels. Conclusions— The CuVIC trial (Effect of Cholesterol Absorption Inhibitor Usage on Target Vessel Dysfunction after Coronary Stenting) has shown that ezetimibe with statins, compared with statin monotherapy, improves functional prognoses, ameliorating endothelial dysfunction in stented coronary arteries, and was associated with larger decreases in oxysterol levels.