Myriocin-mediated up-regulation of hepatocyte apoA-I synthesis is associated with ERK inhibition

Sphingolipids including sphingomyelin have been implicated as potential atherogenic lipids. Studies in apoE (apolipoprotein E)-null mice have revealed that the serine palmitoyltransferase inhibitor myriocin reduces plasma levels of sphingomyelin, ceramide, sphingosine-I-phosphate and glycosphingolipids and that this is associated with potent inhibition of atherosclerosis. Interestingly, hepatic apoA-I (apolipoprotein A-I) synthesis and plasma HDL (high-density lipoprotein)cholesterol levels were also increased in apoE-null mice treated with myriocin. Since myriocin is a known inhibitor of ERK (extracellular-signal-related kinase) phosphorylation, we assessed the possibility that myriocin may be acting to increase hepatic apoA-I production via this pathway. To address this, HepG2 cells and primary mouse hepatocytes were treated with 200 mu M myriocin for up to 48 h. Myriocin increased apoA-I mRNA and protein levels by approx. 3- and 2-fold respectively. Myriocin also increased apoA-I secretion up to 3.5-fold and decreased ERK phosphorylation by approx. 70%. Similar findings were obtained when primary hepatocytes were isolated from apoE-null mice that were treated with myriocin (intraperitoneal injection at a dose of 0.3 mg/kg body weight). Further experiments revealed that the MEK (mitogen-activated protein kinase/ERK kinase) inhibitor PD98059 potently inhibited ERK phosphorylation, as expected, and increased primary hepatocyte apoA-I production by 3-fold. These results indicate that ERK phosphorylation plays a role in regulating hepatic apoA-I expression and suggest that the anti-atherogenic mechanism of action for myriocin may be linked to this pathway

Quantitation of ATP-binding cassette subfamily-A transporter gene expression in primary human brain cells

Five ATP-binding cassette (ABC) subfamily-A transporters (ABCA1, ABCA2, ABCA3, ABCA7 and ABCA8) are expressed in the brain. These transporters may regulate brain lipid transport; however, their relative expression level in isolated human brain cells is unknown. We developed real-time polymerase chain reaction assays to quantify the expression of these genes in human neurons, astrocytes, oligodendrocytes, microglia and cell lines. Neurons expressed predominantly ABCA1 and ABCA3; astrocytes ABCA1, ABCA2 and ABCA3; microglia ABCA1 and oligodendrocytes ABCA2 and ABCA3. Although ABCA7 and ABCA8 expression was relatively low in all cells, the highest expression occurred in microglia and neurons, respectively. ABCA gene expression in the NTERA-2 and MO3.13 cell lines closely resembled the ABCA expression pattern of primary neurons and oligodendrocytes, respectively. © 2006 Lippincott Williams & Wilkins

Reduction of plasma glycosphingolipid levels has no impact on atherosclerosis in apolipoprotein E-null mice

Glycosphingolipids (GSLs) have been implicated as potential atherogenic lipids. Studies in apolipoprotein E-null (apoE-/-) mice indicate that exacerbated tissue GSL accumulation resulting from a-galactosidase deficiency promotes atherosclerosis, whereas the serine palmitoyl transferase inhibitor myriocin (which reduces plasma and tissue levels of several sphingolipids, including sphingomyelin, ceramide, sphingosine-1-phosphate, and GSLs) inhibits atherosclerosis. It is not clear whether GSL synthesis inhibition per se has an impact on atherosclerosis. To address this issue, apoE-/- mice maintained on a high-fat diet were treated with a potent glucosylceramide synthesis inhibitor, D-threo-1-ethylendioxyphenyl-2-palmitoylamino-3-pyrrolidino-propanol (EtDO-P4), 10 mg/kg/day for 94 days, and lesion development was compared in mice that were treated with vehicle only. EtDO-P4 reduced plasma GSL concentration by approximately 50% but did not affect cholesterol or triglyceride levels. Assessment of atherosclerotic lesions at four different sites indicated that EtDO-P4 had no significant impact on lesion area. Thus, despite the previously observed positive correlations between plasma and aortic GSL concentrations and the development of atherosclerosis, and the in vitro evidence implying that GSLs may be pro-atherogenic, our current data indicate that inhibition of GSL synthesis does not inhibit atherosclerosis in vivo. Copyright © 2008 by the American Society for Biochemistry and Molecular Biology, Inc

Myriocin slows the progression of established atherosclerotic lesions in apolipoprotein E gene knockout mice

The serine palmitoyl transferase inhibitor myriocin potently suppresses the development of atherosclerosis in apolipoprotein E (apoE) gene knockout (apoE-/-) mice fed a high-fat diet. This is associated with reduced plasma sphingomyelin (SM) and glycosphingolipid levels. Furthermore, oral administration of myriocin decreases plasma cholesterol and triglyceride (TG) levels. Here, we aimed to determine whether myriocin could inhibit the progression (or stimulate the regression) of established atherosclerotic lesions and to examine potential changes in hepatic and plasma lipid concentrations. Adult apoE-/- mice were fed a high-fat diet for 30 days, and lesion formation was histologically confirmed. Replicate groups of mice were then transferred to either regular chow or chow containing myriocin (0.3 mg/kg/day) and maintained for a further 60 days. Myriocin significantly inhibited the progression of established atherosclerosis when combined lesion areas (aortic sinus, arch, and celiac branch point) were measured. Although the inhibition of lesion progression was observed mainly in the distal regions of the aorta, regression of lesion size was not detected. The inhibition of lesion progression was associated with reductions in hepatic and plasma SM, cholesterol, and TG levels and increased hepatic and plasma apoA-I levels, indicating that the modulation of pathways associated with several classes of atherogenic lipids may be involved. Copyright ©2008 by the American Society for Biochemistry and Molecular Biology, Inc

Glycosphingolipid accumulation inhibits cholesterol efflux via the ABCA1/apolipoprotein A-I pathway: 1-Phenyl-2-decanoylamino-3-morpholino-1- propanol is a novel cholesterol efflux accelerator

Cellular glycosphingolipid (GSL) storage is known to promote cholesterol accumulation. Although physical interactions between GSLs and cholesterol are thought to cause intracellular cholesterol trapping, it is not known whether cholesterol homeostatic mechanisms are also impaired under these conditions. ApoA-I-mediated cholesterol efflux via ABCA1 (ATP-binding cassette transporter A1) is a key regulator of cellular cholesterol balance. Here, we show that apoA-I-mediated cholesterol efflux was inhibited (by up to 53% over 8 h) when fibroblasts were treated with lactosylceramide or the glucocerebrosidase inhibitor conduritol B epoxide. Furthermore, apoA-I-mediated cholesterol efflux from fibroblasts derived from patients with genetic GSL storage diseases (Fabry disease, Sandhoff disease, and GM1 gangliosidosis) was impaired compared with control cells. Conversely, apoA-I-mediated cholesterol efflux from fibroblasts and cholesterol-loaded macrophage foam cells was dose-dependently stimulated (by up to 6-fold over 8 h) by the GSL synthesis inhibitor 1-phenyl-2-decanoylamino- 3-morpholino-1-propanol (PDMP). Unexpectedly, a structurally unrelated GSL synthesis inhibitor, N-butyldeoxynojirimycin, was unable to stimulate apoA-I-mediated cholesterol efflux despite achieving similar GSL depletion. PDMP was found to up-regulate ABCA1 mRNA and protein expression, thereby identifying a contributing mechanism for the observed acceleration of cholesterol efflux to apoA-I. This study reveals a novel defect in cellular cholesterol homeostasis induced by GSL storage and identifies PDMP as a new agent for enhancing cholesterol efflux via the ABCA1/apoA-I pathway. © 2005 by The American Society for Biochemistry and Molecular Biology, Inc

Inhibition of atherosclerosis by the serine palmitoyl transferase inhibitor myriocin is associated with reduced plasma glycosphingolipid concentration

Glycosphingolipids (GSL) have been implicated as potential atherogenic lipids. Inhibition of hepatic serine palmitoyl transferase (SPT) reduces plasma sphingomyelin (SM) levels in the absence of changes in cholesterol or triglyceride (TG) concentration and this leads to a reduction of atherosclerosis in apolipoprotein-E gene knockout (apoE-/-) mice. The possibility that the reduced atherosclerosis resulting from SPT inhibition is associated with decreases in plasma GSL concentration has not been examined and was the primary aim of this investigation. We show that intraperitoneal delivery of the SPT inhibitor myriocin for 9 weeks inhibits atherosclerosis in apoE-/- mice fed a high fat diet. Lesion inhibition was most pronounced at the aortic arch and distal sites of the thoracic and abdominal aorta. There was also a trend towards a reduction in lesion area at the aortic root. Myriocin treatment resulted in significant reductions in both plasma SM and GSL concentration of 42% and 25%, as assessed by enzymatic and HPLC methods, respectively. Moreover, SM and GSL concentrations were significantly correlated, indicating that SPT inhibition suppresses the synthesis of both these sphingolipids concomitantly. The inhibition of atherosclerosis induced by myriocin was not associated with changes in plasma cholesterol or TG concentrations or lipoprotein profiles as determined by FPLC. These data indicate that therapeutic reduction of plasma SM and/or GSL concentrations may offer a novel treatment for atherosclerosis. © 2006 Elsevier Inc. All rights reserved