Glucosylceramide synthase inhibitors D-PDMP and D-EtDO-P4 decrease the GM3 ganglioside level, differ in their effects on insulin receptor autophosphorylation but increase Akt1 kinase phosphorylation in human hepatoma HepG2 cells.

Abstract

Gangliosides function as modulators of several cell growth related receptors. It was shown for caveolin-rich adipocytes, that GM3 ganglioside binds to insulin receptor (IR), dissociates its complex with caveolin, and thus lowers IR autophosphorylation following insulin treatment. We extended those studies into human hepatocyte-derived HepG2 cells, characterized by a high level of IR but low of caveolin. To lower the glycosphingolipid content, estimated by GM3 concentration, two glucosylceramide synthase inhibitors d-threo-1-pheny-2-decanoylamino-3-morpholino-1-propanol (d-PDMP) and d-threo-1-(3,4,-ethylenedioxy)phenyl-2-palmitoylamino-3-pyrrolidino-1-propanol (d-EtDO-P4) were used. d-PDMP at 40 µM or d-EtDO-P4 at 1 µM concentrations in culture medium decreased the GM3 content to 22.3% (17.8-26.1%) and 18.1% (13.7-24.4%), respectively, of the control value. The reduction of GM3 obtained with d-PDMP was accompanied by a 185.1% (153.5-423.8%) significant increase in the level of IR autophosphorylation following cell stimulation with 100 nM insulin. The effect of d-EtDO-P4 on IR autophosphorylation was smaller amounting to an increase by 134.8% (111.3-167.8%) of the control level and statistically non-significant. The effects of d-PDMP and d-EtDO-P4 could also be detected at the level of Akt1 kinase. In cells grown in the presence of d-PDMP the level of phosphorylated Akt1 was 286.0% (151.4%-621.1%) of that in the control. In this case the effect of d-EtDO-P4 was similar: 223.0% (181.4-315.4%) significant increase in phosphorylated Akt1. We assume that glycosphingolipid depletion in HepG2 cells may affect not only IR autophosphorylation but also, independently, the phosphorylation of Akt1, by modifying the membrane microenvironment of this kinase.

Small molecule inhibitors of mammalian glycosylation

“…(+)D- threo -1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP, the active isomer of the four possible isomers) is a glucosylceramide analogue that inhibits glycosphingolipids in cultured cells at 50 μM [263] , [264] . Owing to the role of glycosphingolipids in the aetiology of atherosclerosis, D-PDMP has attracted attention as a potential therapeutic candidate for this disease.…”

Enhancing the sialylation of recombinant EPO produced in CHO cells via the inhibition of glycosphingolipid biosynthesis

“…1). EtDO-P4 (D- threo -ethylenedioxyphenyl-2-palmitoylamino-3-pyrrolidino-propanol) inhibits CGT and is, therefore, capable of reducing GSL biosynthesis in various cells 19,20 . After treating rhEPO-producing EC2-1H9 CHO cells with EtDO-P4, we measured their GSL biosynthesis using thin-layer chromatography (TLC) as previously described 21 .…”

Inhibition of Ganglioside Synthesis Suppressed Liver Cancer Cell Proliferation through Targeting Kinetochore Metaphase Signaling

“…In addition, PDMP is a classic glucosylceramide synthase and lactosylceramide synthase inhibitor targeting beta-1,4-galactosyltransferase 5/6 (B4GALT5/6), which is critical in regulating the biosynthesis of lactosylceramide, a common precursor of lactose series of glycosphingolipids, including gangliosides [60]. PDMP prevented the conversion of ceramide into glycosylated ceramides such as glucosylceramide [61], lactosylceramide [61], monosialo gangliosides GM1 [62] and GM3 [63,64], and disialo ganglioside [61], while induced the content of ceramide [61,65,66] or had limited effects on its conversion into sphingomyelin [67,68] and sphingosine [69]. Ceramide as the central molecule of sphingolipid metabolism has been reported to play a pro-apoptotic and anti-proliferative role in chronic liver diseases such as NASH [70], alcoholic steatohepatitis [71], and hepatic ischemia reperfusion injury [59].…”