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

The potential of sphingolipid depletion for the treatment of atherosclerosis

Sphingolipids have been implicated as potential atherogenic lipids. Inhibition ofhepatic serine palmitoyl transferase (SPT) reduces plasma sphingomyelin (SM) levelsleading to a reduction of atherosclerosis in apolipoprotein-E gene knockout (apoE-/-)mice. In my thesis I have investigated the possibility that the reduced atherosclerosisresulting from SPT inhibition is associated with decreases in plasmaglycosphingolipids (GSL). Furthermore, I examined whether SPT inhibition can leadto regression of atherosclerotic lesions. This thesis shows the SPT inhibitor myriocininhibits atherosclerosis in apoE-/- mice fed a high fat diet. Lesion inhibition was mostpronounced 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. Myriocintreatment resulted in significant reductions in both plasma SM and GSLconcentrations. Moreover, it was demonstrated that myriocin significantly inhibitedthe progression of established atherosclerosis. Although the inhibition of lesionprogression was observed mainly in the distal regions of the aorta, regression of lesionsize was not detected. In addition, this thesis demonstrated for the first time thatselective inhibition of GSL synthesis by the GSL synthesis inhibitor, d-threo-1-ethylendioxyphenyl-2-palmitoylamino-3-pyrrolidino-propanol (EtDO-P4) had nosignificant impact on lesion area in apoE-deficient mice. Thus, despite the previouslyobserved positive correlations between plasma and aortic GSL concentrations and thedevelopment of atherosclerosis, this thesis indicates that inhibition of GSL synthesisdoes not inhibit atherosclerosis in vivo. In other studies we assessed the possibilitythat myriocin may also be acting to increase hepatic apoA-I production via theinhibition of ERK phosphorylation. To address this, HepG2 cells and primary mousehepatocytes were treated with myriocin. This significantly increased apoA-I mRNA,and protein levels. It also increased apoA-I secrection, and decreased ERKphosphorylation. These in vitro data indicate that ERK phosphorylation plays a role inmodulating apoA-I expression, and that myriocin’s mechanism of action is linked tothis pathway. Overall, this thesis has expanded the current literature regarding the roleof sphingolipid synthesis inhibition and atherosclerosis

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