Inhibition of Rab prenylation by statins induces cellular glycosphingolipid remodeling

Abstract

Statins, which specifically inhibit HMG Co-A reductase, the rate-limiting step of cholesterol biosynthesis, are widely prescribed to reduce serum cholesterol and cardiac risk, but many other effects are seen. We now show an effect of these drugs to induce profound changes in the step-wise synthesis of glycosphingolipids (GSLs) in the Golgi. Glucosylceramide (GlcCer) was increased several-fold in all cell lines tested, demonstrating a widespread effect. Additionally, de novo or elevated lactotriaosylceramide (Lc3Cer; GlcNAcβ1-3Galβ1-4GlcCer) synthesis was observed in 70%. Western blot showed that GlcCer synthase (GCS) was elevated by statins, and GCS and Lc3Cer synthase (Lc3S) activities were increased; however, transcript was elevated for Lc3S only. Supplementation with the isoprenoid precursor, geranylgeranyl pyrophosphate (GGPP), a downstream product of HMG Co-A reductase, reversed statin-induced glycosyltransferase and GSL elevation. The Rab geranylgeranyl transferase inhibitor 3-PEHPC, but not specific inhibitors of farnesyl transferase, or geranylgeranyl transferase I, was sufficient to replicate statin-induced GlcCer and Lc3Cer synthesis, supporting a Rab prenylation-dependent mechanism. While total cholesterol was unaffected, the trans-Golgi network (TGN) cholesterol pool was dissipated and medial Golgi GCS partially relocated by statins. GSL-dependent vesicular retrograde transport of Verotoxin and cholera toxin to the Golgi/endoplasmic reticulum were blocked after statin or 3-PEHPC treatment, suggesting aberrant, prenylation-dependent vesicular traffic as a basis of glycosyltransferase increase and GSL remodeling. These in vitro studies indicate a previously unreported link between Rab prenylation and regulation of GCS activity and GlcCer metabolism.

Low level of the plasma sphingolipid, glucosylceramide, is associated with thrombotic diseases

“…Plasma samples from the Scripps VTE registry (105 Caucasian VTE patients and 105 age matched controls), 11,12 the Valencia VTE registry (320 VTE Caucasian cases and 316 Caucasian controls), 13 and one MI cohort (209 MI patients from Genetic and Viral Attributes of Myocardial Infarction study and 197 healthy controls from Genetic Attributes of Thrombosis Epidemiology study) 14,15 were studied. The subjects taking lipid lowering medications including statins were excluded to avoid any interaction of dynamic lipid metabolism including glycolipids 11,16 for VTE studies. Further, to address the GlcCer independent association with VTE for known risk factors of VTE, subjects with known thrombophilic defects, including factor V Leiden and prothrombin G20210A mutations and anyone currently taking oral anticoagulants or oral contraceptives were excluded in the Valencia VTE study.…”

Medicines and Vegetable Oils as Hidden Causes of Cardiovascular Disease and Diabetes

“…Rab is activated by prenylation and plays an essential role in membrane trafficking of the insulin receptor and other proteins. Inhibition of dolichol synthesis and Rab prenylation by statins leads to cellular glycosphingolipid remodeling [12] . Other aspects of statins to stimulate the onset of DM have been reviewed by Brault et al [13] .…”

Synthesis of a novel photoactivatable glucosylceramide cross-linker

“…Near-confluent DU145 cells, maintained with MEM supplemented with 10% FBS, were washed with D-PBS, detached by trypsin, collected in an equal volume of FBS to inhibit trypsin, and washed twice with ice-cold D-PBS (360 g, 4°C), and pellets were stored at 80°C. Cell pellets were suspended in one volume of ice-cold homogenization buffer (10 mM Tris-HCl pH 7.4, 10 mM KCl, 1.5 mM MgCl 2 , 0.5 M sucrose), and homogenized with GlcCer synthesis and trafficking are, in addition, regulated by statin-sensitive prenylation mechanisms (17).…”