Regulation of glycosylation. Three enzymes compete for a common pool of dolichyl phosphate in vivo

We examined changes in the levels of the dolichol forms in Chinese hamster ovary cells containing alterations in the levels of activity of two enzymes in the oligosaccharyl-P-P-dolichol biosynthetic pathway, namely UDP-GlcNAc:dolichyl phosphate:GlcNAc-phosphotransferase (GlcNAc-1-phosphotransferase) and mannosylphosphoryldolichol (Man-P-Dol) synthase. Under normal conditions in wild type cells, Glc3Man9GlcNAc2-pyrophosphoryldolichol was the most abundant form. Of the other anionic forms of dolichols, dolichyl phosphate, Man-P-Dol, glucosylphosphoryldolichol, and Man5GlcNAc2-pyrophosphoryl dolichol were approximately equally abundant. When 3E11 cells (a tunicamycin-resistant Chinese hamster ovary line containing 15 times more GlcNAc-1-phosphotransferase activity than wild type), B4-2-1 cells (a mutant lacking Man-P-Dol synthase activity), and wild type cells incubated with or without tunicamycin were utilized, significant changes in the levels of most of the anionic dolichol derivatives, with the exception of dolichyl phosphate, were found. Since changes in dolichyl phosphate levels were not detected under a variety of conditions where the levels of enzyme activity utilizing this substrate were varied, all three enzymes appear to have access to the same pool of dolichyl phosphate, and further, to have similar Km values for dolichyl phosphate.     

A Chinese hamster ovary cell mutant F2A8 utilizes polyprenol rather than dolichol for its lipid-dependent asparagine-linked glycosylation reactions

Previous results suggested that F2A8, a glycosylation mutant of Chinese hamster ovary cells, had a lower amount of dolichyl phosphate available for asparagine-linked glycosylation reactions relative to parental cells. The steady-state amounts and identities of polyisoprenoid lipids were determined by incubating F2A8, its parental cell line B4-2-1, and wild-type Chinese hamster ovary cells for 24 h with [2-3H]mevalonate. The neutral lipids, ubiquinone, cholesterol, and cholesteryl esters, which were the most highly labeled from [3H]mevalonate, were labeled equally in all three cell types. In wild-type and B4-2-1 cells, mevalonate incorporation into the anionic glycosylated and phosphorylated derivatives of dolichol was 10-fold higher than into the neutral free dolichol and dolichyl esters. In contrast, in F2A8 cells, label accumulated in neutral polyisoprenol lipids, so that the ratio of neutral to anionic lipids was 1:1 rather than 1:10. In wild-type and B4-2-1 cells, the polyisoprenoid found as free alcohol and in phosphorylated and glycosylated forms was shown by high pressure liquid chromatography using a silica column to be primarily dolichol, a polyisoprenol that has a saturated terminal isoprene unit. In contrast, in F2A8 cells the polyisoprenoid found primarily as the free alcohol and in phosphorylated and glycosylated forms appeared to be completely unsaturated polyprenol. The distribution of chain lengths of the labeled polyisoprenols of F2A8, B4-2-1, and wild-type cells was the same as determined by high pressure liquid chromatography using a reverse-phase column, with the predominant chain length being 19 isoprene units. These results combined with our previous studies on the phenotype of the F2A8 mutant indicate that the unsaturated polyprenyl phosphate derivatives do not function as well as dolichyl phosphate derivatives in cellular glycosylation reactions.     

Substrate specificity of N-acetylglucosamine 1-phosphate transferase activity in Chinese hamster ovary cells.

The assembly pathway of the oligosaccharide chains of asparagine-linked glycoproteins in mammalian cells begins with the formation of GlcNAc-PP-dolichol in a reaction catalysed by the enzyme N-acetylglucosamine 1-phosphate transferase. We have investigated the efficiency of two lipid substrates for the transferase activity in an in vitro assay using Chinese hamster ovary (CHO) cell membranes as an enzyme source. Experiments were carried out with varying concentrations of dolichyl phosphate or its precursor, polyprenyl phosphate. We determined that enzyme activity was optimal at pH 9, where the enzyme exhibited a 3-fold higher Vmax and a 2-fold lower Km for the dolichol substrate. At pH 7.4, the Km and Vmax differences between the two lipids were 10-fold. Under all assay conditions tested, we found that GlcNAc-PP-lipid was the only product formed. We conclude from these results that dolichyl phosphate rather than polyprenyl phosphate is the preferred substrate for the transferase enzyme in CHO cells. This observation is significant in light of the fact that we have previously isolated CHO glycosylation mutants which fail to convert polyprenol into dolichol, and hence utilize polyprenyl derivatives for glycosylation reactions. Thus, these results contribute to our understanding of the glycosylation defects in the mutant cell lines.     

A single enzyme catalyzes the synthesis of the mannosylphosphoryl derivative of dolichol and retinol in rat liver and Chinese hamster ovary cells

It is well established that mannosylphosphoryldolichol participates in the synthesis of N-linked glycoproteins by donating mannosyl residues to oligosaccharide-lipid intermediates. It has been suggested that mannosylphosphorylretinol also is involved in glycoprotein biosynthesis. We conclude that one synthase catalyzes the synthesis of both mannosylphosphoryldolichol and mannosylphosphorylretinol in rat liver tissue and Chinese hamster ovary cells, based on the following results. 1) The enzyme in rat liver microsomes that synthesizes mannosylphosphoryldolichol and mannosylphosphorylretinol is inactivated at the same rate at 55 degrees C. 2) In membranes of both rat liver and Chinese hamster ovary cells, exogenous dolichyl phosphate and retinyl phosphate compete with each other for mannosyl-lipid synthesis. However, in both systems adding exogenous retinyl phosphate has no effect on the synthesis of mannosylphosphoryldolichol from endogenous dolichyl phosphate in the membranes. 3) Membranes prepared from a mutant of Chinese hamster ovary cells which is devoid of mannosylphosphoryldolichol synthase lack the ability to synthesize mannosylphosphorylretinol.     

Lec9 CHO glycosylation mutants are defective in the synthesis of dolichol

Lec9 Chinese hamster ovary cells were found previously to be defective in the synthesis of N-linked glycans. This appeared to be the result of a defect in the synthesis of oligosaccharide lipid and lipid phosphate (Rosenwald, Stanley, and Krag. 1989. Mol. Cell. Biol. 9: 914-924). In this study we analyzed the steady state levels of long-chain polyisoprenyl lipids in Lec9 cells. We found that Lec9 cells are defective in the synthesis of dolichol. They accumulated a presumed precursor to dolichol, cis-a-unsaturated polyprenol and used this lipid in the synthesis of oligosaccharide lipid. Chain lengths of the activated polyprenols in Lec9 were the same lengths as dolichols in parental cells. Lec9 cells had increased levels of monosaccharylphosphoryl lipid and decreased levels of oligosaccharylpyrophosphoryl lipid compared to parental cells. The defect in Lec9 cells was specific for dolichol synthesis, since other aspects of [3H]mevalonate metabolism in Lec9 cells were the same as in parental cells. We hypothesize that Lec9 cells are defective in polyprenol reductase activity.     

A mutant of Chinese hamster ovary cells with a reduction in levels of dolichyl phosphate available for glycosylation

F2A8, a glycosylation mutant of Chinese hamster ovary cells, was isolated without prior enrichment or selective procedures by screening colonies for reduced [3H]mannose incorporation into macromolecules. F2A8 cells incubated with [3H]mannose synthesized 70% the amount of labeled GDP-mannose found in parental cells, and the same oligosaccharides attached to lipid and protein as did parental cells, but in reduced amounts. Incorporation of radioactivity from labeled mannose into saccharide-lipids and into total glycopeptides of F2A8 was reduced 7-fold compared to parental cells. In addition, glycosylation of the vesicular stomatitis virus glycoprotein was reduced in F2A8 cells as assessed by a mobility intermediate between normally glycosylated and unglycosylated protein during sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In vitro assays using membrane preparations showed that F2A8 had parental levels of glucosylphosphoryldolichol synthase and of UDP-GlcNAc:dolichyl phosphate:GlcNAc-phosphotransferase when the enzymatic determinations were done in the presence of exogenous dolichyl phosphate. However, 5-fold less glucosylphosphoryldolichol synthase activity was detected in membranes of F2A8 compared to membranes of parental cells in assays relying on endogenous lipid substrate. F2A8 appears to have reduced amounts of dolichyl phosphate available for its glycosylation reactions.     

Changes in the Levels of Dolichol and Dolichyl Phosphate in a Murine Model of Niemann-Pick''s Type C Disease

Abstract: The distributions of mevalonate pathway lipids in various organs of a mouse strain used as a model for Niemann-Pick's type C disease were analyzed. Extensive accumulation of cholesterol was observed in all tissues with the exception of the brain, where the content of this lipid was decreased. The changes in total dolichol contents of most organs varied from a 50% decrease in the lung to a twofold increase in kidney and heart. There was relative enrichment of longer-chain dolichols, but no increase in the relative amount of α-unsaturated polyprenols was observed. The levels of dolichyl phosphate in all organs were increased, and most of this lipid was associated with bound oligosaccharides or proteins. Ubiquinone levels were largely unchanged. Subfractionation studies revealed that heavy and light lysosomes exhibited a 10-fold increase in cholesterol level, the amount of dolichol was decreased in lysosomes and increased in microsomes, and there was an increase in the dolichyl phosphate levels of all three of these subfractions. These results indicate that in diseased mice cholesterol accumulation in various organs is paralleled by an increase in the dolichyl phosphate concentration, whereas dolichol transport from the endoplasmic reticulum to lysosomes is inhibited.     

Submicrosomal distribution of dolichol kinase and dolichyl phosphate phosphatase in the microsomes of germinating soybeans

The availability of dolichyl phosphate is a major factor in the rate of formation of N-linked glycoproteins in mammalian cells. Recent studies in our laboratory suggested that glycoproteins required for seed germination and early plant development are formed via the dolichyl phosphate pathway. Soybean microsomes contain dolichol kinase and dolichyl phosphate phosphatase, enzymes that regulate dolichyl phosphate levels by interconversion of dolichyl phosphate and dolichol. In the present study, soybean microsomes were fractionated into rough and smooth endoplasmic reticulum and Golgi, and the activities of dolichol kinase and dolichyl phosphate phosphatase were measured in each. Submicrosomal fractions were obtained using a procedure developed for rat liver, and were characterized by marker enzymes, RNA content and electron microscopy. The site of N-glycosylation, the rough endoplasmic reticulum, contained high levels of both dolichol kinase and dolichyl phosphate phosphatase. This makes possible a mechanism whereby glycoprotein formation during seed germination is regulated by availability of dolichyl phosphate.     

Increased synthesis and concentration of dolichyl phosphate in mouse spleens during phenylhydrazine-induced erythropoiesis

Erythropoietic spleens from mice treated with phenylhydrazine synthesized dolichol + dolichyl acyl esters at a higher rate than did normal spleens, and this increased synthesis occurred 1–2 days after the peak of cholesterol synthesis. We have further characterized this dolichol synthesis and have found that at 4 days following phenylhydrazine treatment, dolichyl phosphate accounted for 30% of total synthesis, and at this time 60% of tissue dolichol was phosphorylated. In contrast, treatment with erythropoietin caused simultaneous increases in dolichol and cholesterol synthesis, with very low levels of dolichyl phosphate synthesis. The present results show that the synthesis and the mass of dolichyl phosphate increased in the spleens of phenyl-hydrazine- but not erythropoietin- treated mice.     

Separation and purification of dolichol and dolichyl phosphate by anion-exchange paper chromatography: application to cultured cells.

Dolichyl phosphate, dolichol C80-105 (dolichol 17:dihydroheptadecaprenol-dolichol 21:dihydrohexeicosaprenol), and dolichol C55 (dolichol 11:dihydroundecaprenol) were separated by anion-exchange paper chromatography. Squalene, sterols, phospholipids, anionic glycolipids, and glycerol did not migrate as dolichyl phosphate, dolichol C80-105, and dolichol C55 under our elution conditions. However, since the Rf of triglycerides was similar to that of dolichol C80-105, saponification, prior to chromatography, removed traces of triglycerides. Silica gel thin-layer chromatography (TLC) allowed the separation of dolichol C80-105 from dolichol C55, whereas dolichyl phosphate was eluted with other lipids. Incubation of spontaneously transformed cells derived from rat astrocytes primary cultures with [2-14C]acetate, saponification of the extracted lipids, and anion-exchange paper chromatography revealed the presence of radioactive dolichyl phosphate and dolichol C80-105 (15 pmol/mg protein). Extraction of labeled dolichyl phosphate followed by acid phosphatase treatment and subsequent analysis on TLC confirmed the identity of dolichyl phosphate since all the radioactivity was associated with dolichol C55. Treatment of the transformed cells with 30 microM 7-ketocholesterol or 7 beta-hydroxycholesterol stimulated markedly (two- to threefold) the incorporation of [2-14C]-acetate in both dolichol C80-105 and dolichyl phosphate. These data demonstrate that anion-exchange paper chromatography is technically suitable for the separation and analysis of dolichol C55, dolichol C80-105, and dolichyl phosphate in cultured cells prelabeled with radioactive precursors.     

Effects of peroxisome proliferators gemfibrozil and clofibrate on syntheses of dolichol and cholesterol in rat liver

The effects of two peroxisome proliferators, gemfibrozil and clofibrate, on syntheses of dolichol and cholesterol in rat liver were investigated. Gemfibrozil did not affect the overall content of dolichyl phosphate, but it changed the chain-length distribution of dolichyl phosphate, increasing the levels of species with shorter isoprene units. Gemfibrozil suppressed synthesis of dolichyl phosphate from [(3)H]mevalonate and [(3)H]farnesyl pyrophosphate in rat liver. In contrast, clofibrate increased the content of dolichol (free and acyl ester forms). It remarkably enhanced dolichol synthesis from mevalonate, but did not affect dolichol synthesis from farnesyl pyrophosphate. Gemfibrozil elevated cholesterol synthesis from [(14)C]acetate, but did not affect the synthesis from mevalonate. Clofibrate suppressed cholesterol synthesis from acetate, but did not affect cholesterol synthesis from mevalonate. These results suggest that gemfibrozil suppresses synthesis of dolichyl phosphate by inhibiting, at the least, the pathway from farnesyl pyrophosphate to dolichyl phosphate. As a result, the chain-length pattern of dolichyl phosphate may show an increase in shorter isoprene units. Clofibrate may increase the content of dolichol by enhancing dolichol synthesis from mevalonate. Gemfibrozil may increase cholesterol synthesis by activating the pathway from acetate to mevalonate. Unlike gemfibrozil, clofibrate may decrease cholesterol synthesis by inhibiting the pathway from acetate to mevalonate.     

Relationships among dolichyl phosphate, glycoprotein synthesis, and cell culture growth

Following treatment of Chinese hamster ovary cells with inhibitors of mevalonate biosynthesis in the presence of exogenous cholesterol, the cellular concentration of phosphorylated dolichol and the incorporation of [3H]mannose into dolichol-linked saccharides and N-linked glycoproteins declined coincident with a decline in DNA synthesis. Addition of mevalonate to the culture medium increased rates of mannose incorporation into lipid-linked saccharides and restored mannose incorporation into N-linked glycoproteins to control levels within 4 h. After an additional 4 h, synchronized DNA synthesis began. Inhibition of the synthesis of lipid-linked oligosaccharides and N-linked glycoproteins by tunicamycin prevented the induction of DNA synthesis by mevalonate, indicating that glycoprotein synthesis was required for cell division. The results suggest that the rate of cell culture growth may be influenced by the level of dolichyl phosphate acting to limit the synthesis of N-linked glycoproteins.     

Genomic organization and expression of hamster UDP-N-acetylglucosarmine:dolichyl phosphate N-acetylglucosaminyl phosphoryl transferase

The hamster gene for uridine diphosphate N-acetyl-D-glucosamine:dolichyl phosphate N-acetylglucosaminyl phosphoryl transferase(L-G1PT) was found to extend over 6.5 kb and to contain nineexons. The exons ranged in size from 63 to 214 bp, encodingthe 408 amino acid protein. The introns ranged from 85 bp to1.4 kb. Upstream 5' sequences included two possible TATA boxes,one possible CCAAT box and at least two potential GC boxes.Heterologous expression was successful in Schizosaccharomycespombe, and resulted in cells that were tunicamycin resistantand had 12-fold more L-G1PT activity than wild-type cells. Antiserumprepared to a hydrophilic peptide (residues 300–320) ofthe L-G1PT protein reacted with a 35–36 kDa protein inmembrane samples from Chinese hamster ovary (CHO) cells andS.pombe cells that had increased levels of L-G1PT activity.In both cases, antigenic peptide competed with the 35–36kDa protein detected by the antiserum. N-acetylglucosarmine 1-phosphate transferase dolichol glycosylation     

Depression of hepatic dolichol levels by cholesterol feeding

A study was conducted to determine whether repression of 3-hydroxy-3-methylglutaryl CoA reductase by a chronic high-cholesterol diet would deplete hepatic dolichol levels. Four-week-old male C57BL/6J mice were maintained on a control diet or a diet supplemented with 5% cholesterol. Animals from both groups were killed at various times and reductase activity and levels of free dolichol, dolichyl acyl ester, dolichyl phosphate, and ubiquinone were measured. The reductase activity was reduced by 90% within 1 week and remained depressed through 56 days. Initially, the levels of the free dolichol, acyl ester, phosphoryl ester, and ubiquinone were 7, 16, 5, and 80 micrograms/g liver, respectively. Early increases in the concentration of dolichyl phosphate and free dolichol were similar in both the cholesterol-fed and control groups. However, in the cholesterol-fed group the concentration of dolichyl acyl esters was only 50% of that in the control group by 7 days and it remained lower throughout the experiment. Total dolichol levels were lower by about 30%. Ubiquinone levels were transiently depressed at 7 days by 33% but returned to control levels by 4 weeks. After 56 days, the control values of dolichol and dolichyl phosphate remained constant whereas the dolichyl acyl ester levels continuously increased to a value of 133 micrograms/g of liver by 156 days. Subcellular fractionation of livers from 4-week-old mice indicated a lysosomal distribution of dolichol and dolichyl acyl ester and a lysosomal and microsomal distribution of dolichyl phosphate.     

Cell-cycle dependence of dolichyl phosphate biosynthesis

The cell-cycle dependence of dolichyl phosphate biosynthesis has been investigated in mouse L-1210 cells fractionated by centrifugal elutriation. Dolichyl phosphate levels increased linearly through the cell cycle, reaching a value in late S phase twice that of early G1. The cell-cycle dependences of four dolichyl phosphate metabolizing enzymes have been measured: cis-prenyltransferase, CTP-dependent dolichol kinase, dolichyl phosphatase, and dolichyl pyrophosphatase. The kinase, the cis-prenyltransferase, and the pyrophosphatase showed cell-cycle variations, increasing through G1 to a maximum in S phase while the monophosphatase activity was cell-cycle independent. The rate of accumulation of dolichyl phosphate was not affected by growing the cells in mevalonolactone-supplemented media. The evidence presented is consistent with models in which either the cis-prenyltransferase or the kinase/phosphatase couple (or both) regulates the levels of dolichyl phosphate in the cell.     

Metabolic interconversion of dolichol and dolichyl phosphate during development of the sea urchin embryo

The in vivo and in vitro synthesis and turnover of dolichol and dolichyl phosphate have been studied over the course of early development in sea urchin embryos. Synthesis of dolichol and dolichyl phosphate was studied in vivo and in vitro using [3H]acetate and [14C] isopentenylpyrophosphate, respectively, as precursors. Both the in vivo and in vitro results indicate that the principal labeled end product of de novo synthesis is the free alcohol, and that this alcohol is subsequently phosphorylated to produce dolichyl phosphate. The presence of 30 microM compactin inhibits the de novo synthesis of dolichol from [3H]acetate by greater than 90%, but has no effect on the incorporation of 32Pi into dolichyl phosphate for more than 6 h, thus suggesting that during this time interval the major source of dolichyl phosphate is preformed dolichol. The rate of turnover of the [3H]acetate-labeled polyisoprenoid backbone of dolichyl phosphate is very slow (t1/2 = 40-70 h). In contrast, the rate of loss of the [32P]phosphate headgroup is more rapid (t1/2 = 5.7-7.7 h) and increases over the course of development. Finally, dolichyl phosphate phosphatase activity has been measured in vitro. The activity of this enzyme, which can be distinguished from phosphatidic acid phosphatase, was found to increase as a function of development, in qualitative agreement with the increased turnover of 32P from dolichyl phosphate observed in vivo. These results suggest that the phosphate moiety of dolichyl phosphate is in a dynamic state, and that dolichol kinase and dolichyl phosphate phosphatase play key roles in regulating the cellular level of dolichyl phosphate.     

The influence of dolichol, dolichol esters, and dolichyl phosphate on phospholipid polymorphism and fluidity in model membranes

The effect of dolichols, polyprenols, dolichol esterified with fatty acids, and dolichyl phosphate on the structure and fluidity of model membranes was studied using 31P NMR, small-angle x-ray scattering, differential scanning calorimetry, and freeze-fracture electron microscopy. These studies suggest that dolichol and dolichol derivatives destabilize unsaturated phosphatidylethanolamine containing bilayer structures and promote hexagonal II phase formation; high concentrations of dolichol induce lipid structures characterized by "isotropic" 31P NMR and particulate fracture faces; dolichol, contrary to cholesterol, has no effect on the thermotropic behavior of membranes consisting of phosphatidylcholine, while dolichyl-P incorporation abolishes the transition from the gel to liquid crystalline phase in 1,2-dimyristoyl-sn-glycero-3-phosphocholine; both dolichol and dolichyl-P increase the fatty acid fluidity in phosphatidylethanolamine mixtures; the effect of dolichol on bilayer structure and fluidity is more pronounced with increasing number of isoprene residues; dolichol esters are only soluble to a limited extent in the bilayer and segregates into domains at low concentrations; the results are consistent with a localization of dolichyl-P in which the phosphate group is oriented to the water interphase. The induction of hexagonal II phase by dolichyl-P may elicit the transmembrane movement of glycosylated lipid intermediate.     

Dihydroheptaprenyl and dihydrodecaprenyl monophosphates induce apoptosis mediated by activation of caspase-3-like protease

Dolichyl phosphate, an essential carrier lipid in the biosynthesis of N-linked glycoprotein, has been found to induce apoptosis in rat glioma C6 cells and human monoblastic leukemia U937 cells. In the present study, dolichyl phosphate and structurally related compounds were examined regarding their apoptosis-inducing activities in U937 cells. Dihydroheptaprenyl and dihydrodecaprenyl phosphates, of which isoprene units are shorter than that of dolichyl phosphate, induced apoptosis in U937 cells. This phenomenon occurred in a dose- and time-dependent manner, as seen with dolichyl phosphate-induced apoptosis. Derivatives of the same isoprene units of dolichyl phosphate, such as dolichol, dolichal or dolichoic acid, did not induce DNA fragmentation. Farnesyl phosphate and geranylgeranyl phosphate also failed to induce apoptosis. During apoptosis, the caspase family of cysteine proteases play important roles. We observed that apoptosis induced by dihydroprenyl phosphate was mediated by caspase-3-like (CPP32-like) activation but not by caspase-1-like (ICE-like) activation. This caspase-3-like activation was inhibited by a specific inhibitor of caspase-3, DEVD-CHO, but not by an caspase-1 inhibitor YVAD-CHO. We interpret these results to mean that dihydroprenyl phosphates with more than seven isoprene units have apoptosis-inducing activity and that their signal is mediated by caspase-3-like activation.     

Effects of turpentine-induced inflammation on the synthesis of dolichol-linked intermediates of N-glycosylation and the phosphorylation of dolichol by CTP-dependent dolichol kinase

Inflammation was induced in rats by the subcutaneous injection of turpentine. Microsomes were prepared from the livers between 2 and 72 h after injection. Mannose and glucose incorporation into mannosyl and glucosyl dolichyl monophosphate was increased 2-fold over saline-injected controls 24 h after induction of inflammation. Synthesis of glycosylated dolichyl pyrophosphoryl oligosaccharides was also increased compared to controls. Extraction and assay of dolichol monophosphate from inflamed and control rat liver microsomes indicated that the endogenous levels of the lipid were elevated in the inflamed state. CTP-dependent phosphorylation of endogenous dolichol was also found to increase in microsomes from inflamed rats 24 h after injection of turpentine. When exogenous dolichol was added to the microsomal system an increase in phosphorylation was observed as early as 6 h after turpentine injection. Furthermore, the increase appeared to be biphasic, there being two peaks of elevated activity at 12 and 36-48 h after induction of inflammation. The earlier peak was the greater of the two. The results suggest that the increase in glycosylation of dolichol derivatives was due to greater amounts of endogenous dolichol monophosphate. The increase in dolichol monophosphate was itself due to greater availability of dolichol and an increase in the levels of CTP-dependent dolichol kinase.     

Effects of mevinolin treatment on tissue dolichol and ubiquinone levels in the rat.

Rats were treated with mevinolin by intraperitoneal injection (15 days) or dietary administration (30 days). The cholesterol, dolichol, dolichyl phosphate and ubiquinone contents of the liver, brain, heart, muscle and blood were then investigated. The cholesterol contents of these organs did not change significantly, with the exception of muscle. Intraperitoneal administration of the drug increases the amount of dolichol in liver, muscle and blood and decreases the dolichyl-P amount in muscle. The same treatment increases the level of ubiquinone in muscle and blood and decreases this value in liver and heart. Oral administration decreases dolichol, dolichyl-P and ubiquinone levels in heart and muscle, while in liver the dolichol level is elevated and ubiquinone level lowered. In brain the amount of dolichyl-P is increased. Intraperitoneal injection of mevinolin also modifies the liver dolichol and dolichyl-P isoprenoid pattern, with an increase in shorter chain polyisoprenes. The levels of dolichol and ubiquinone in the blood do not follow the changes observed in other tissues. Incorporation of [3H]acetate into cholesterol by liver slices prepared from mevinolin-treated rats exhibited an increase, whereas in brain no change was seen. Labeling of dolichol and ubiquinone was increased in both liver and brain, but incorporation into dolichyl phosphate remained relatively stable. The results indicate that mevinolin affects not only HMG-CoA reductase but, to some extent, also affects certain of the peripheral enzymes, resulting in considerable effects on the various mevalonate pathway lipids.