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.     

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.     

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.     

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.     

Involvement of retinoid X receptor alpha in coenzyme Q metabolism

The nuclear retinoid X receptor alpha (RXRalpha) is the heterodimer partner in several nuclear receptors, some of them regulating lipid biosynthesis. Since coenzyme Q (CoQ) levels are greatly modified in aging and a number of diseases, we have investigated the involvement of RXRalpha in the biosynthetic regulation of this lipid by using a hepatocyte-specific RXRalpha-deficient mouse strain (RXRalpha-def). In the receptor-deficient liver, the amount of CoQ decreased to half of the control, and it was demonstrated that this decrease was caused by a significantly lowered rate of biosynthesis. On the other hand, induction of CoQ was extensive in both control and RXRalpha-def liver using the peroxisomal inducer di(2-ethylhexyl)phthalate (DEHP). Since the RXRalpha deficiency was specific to liver, no change in CoQ content or biosynthesis was observed in kidney. The other mevalonate pathway lipids, cholesterol and dolichol, were unchanged in the RXRalpha-def liver. Upon treatment with DEHP, cholesterol decreased in the control but remained unchanged in the receptor-deficient mice. In control mice, cold exposure elevated CoQ levels by 60%, but this induction did not occur in the liver of RXRalpha-def mice. In contrast, PPARalpha-null mice, which lack induction upon treatment with peroxisomal inducers, respond to cold exposure and CoQ content is increased. The amount of cholesterol decreased in both control and RXRalpha-def liver upon cold treatment. The results demonstrate that RXRalpha is required for CoQ biosynthesis and for its induction upon cold treatment, but does not appear to be involved in the basic synthesis of cholesterol and dolichol. The receptor is not involved in the elevated CoQ biosynthesis during peroxisomal induction.     

Changes of dolichol and dolichyl fatty acyl esters during the germination and development of soybeans.

The amounts of dolichol and dolichyl fatty acyl esters and their composition in various parts of soybean seedlings were determined during germination and development. The dolichol content of cotyledons decreased during germination. Dolichyl fatty acyl esters were identified in cotyledons and the amount was estimated by high performance liquid chromatography. The relative amounts of short-chain dolichols of 15, 16, and 17 isoprene units increased during development of the seedlings. The homologue distribution of free dolichol was different from that of dolichyl fatty acyl esters. The relative amounts of dolichols with 16, 17, and 18 isoprene units were greater in free dolichol than in dolichyl fatty acyl esters. The percentages of long-chain saturated fatty acids in dolichyl fatty acyl esters, specifically 21:0, 22:0, 23:0, 24:0, and 25:0, increased during development. These fatty acids represented more than 40% of the fatty acids in dolichyl fatty acyl esters in stems. These results suggest that dolichyl fatty acyl esters are not a storage form of dolichol. The large accumulation of dolichol and dolichyl fatty acyl esters in the leaves, where photosynthesis takes place, suggests some other function.     

Cholesterol signaling at the endoplasmic reticulum occurs in npc1(-/-) but not in npc1(-/-), LDLR(-/-) mice

It remains controversial whether deficiency of the Niemann-Pick C1 (npc1) protein results in altered cholesterol signaling at the endoplasmic reticulum (ER). In this report, we have measured the processed, nuclear form of sterol regulatory element binding protein (SREBP)-1 in livers of npc1 wild-type, heterozygous, and homozygous deficient mice, alone, and in combination with deficiencies of the low density lipoprotein receptor (LDLR) or the multiple drug resistant (mdr)1a, P-glycoprotein. Cleavage of SREBPs to activated forms normally occurs when the ER is deficient in cholesterol. A large decrease in processed SREBP-1 was evident in fasted npc1(-/-) mice and npc1(-/-), mdr1a(-/-) mice, with no decrease evident in npc1(-/-), LDLR(-/-) mice. These results suggest that the increase in cellular cholesterol which occurs in npc1(-/-) and in npc1(-/-), mdr1a(-/-) mice includes the sites responsible for cholesterol signaling, while the similar increase in cholesterol found in npc1(-/-), LDLR(-/-) mice does not.     

Disturbed cholesterol homeostasis in a peroxisome-deficient PEX2 knockout mouse model

We evaluated the major pathways of cholesterol regulation in the peroxisome-deficient PEX2(-/-) mouse, a model for Zellweger syndrome. Zellweger syndrome is a lethal inherited disorder characterized by severe defects in peroxisome biogenesis and peroxisomal protein import. Compared with wild-type mice, PEX2(-/-) mice have decreased total and high-density lipoprotein cholesterol levels in plasma. Hepatic expression of the SREBP-2 gene is increased 2.5-fold in PEX2(-/-) mice and is associated with increased activities and increased protein and expression levels of SREBP-2-regulated cholesterol biosynthetic enzymes. However, the upregulated cholesterogenic enzymes appear to function with altered efficiency, associated with the loss of peroxisomal compartmentalization. The rate of cholesterol biosynthesis in 7- to 9-day-old PEX2(-/-) mice is markedly increased in most tissues, except in the brain and kidneys, where it is reduced. While the cholesterol content of most tissues is normal in PEX2(-/-) mice, in the knockout mouse liver it is decreased by 40% relative to that in control mice. The classic pathway of bile acid biosynthesis is downregulated in PEX2(-/-) mice. However, expression of CYP27A1, the rate-determining enzyme in the alternate pathway of bile acid synthesis, is upregulated threefold in the PEX2(-/-) mouse liver. The expression of hepatic ATP-binding cassette (ABC) transporters (ABCA1 and ABCG1) involved in cholesterol efflux is not affected in PEX2(-/-) mice. These data illustrate the diversity in cholesterol regulatory responses among different organs in postnatal peroxisome-deficient mice and demonstrate that peroxisomes are critical for maintaining cholesterol homeostasis in the neonatal mouse.     

Lysosomal unesterified cholesterol content correlates with liver cell death in murine Niemann-Pick type C disease

Niemann-Pick type C (NPC) disease is a multisystem disorder resulting from mutations in the NPC1 gene that encodes a protein involved in intracellular cholesterol trafficking. Significant liver dysfunction is frequently seen in patients with this disease. The current studies used npc1 mutant mice to investigate the association between liver dysfunction and unesterified cholesterol accumulation, a hallmark of NPC disease. Data from 92 npc1(-/-) mice (age range, 9-56 days) revealed a significant positive correlation between the plasma activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) and whole liver cholesterol content. In 56 day old npc1(-/-) mice that had been fed from 35 days of age a rodent diet or the same diet containing either cholesterol (1.0%, w/w) or ezetimibe (a sterol absorption inhibitor; 0.0125%, w/w), whole liver cholesterol content averaged 33.5 +/- 1.1, 87.9 +/- 1.7, and 20.8 +/- 0.9 mg, respectively. Again, plasma ALT and AST activities were positively correlated with hepatic cholesterol content. In contrast, plasma transaminase levels remained in the normal range in npc1(+/+) mice, in which hepatic esterified cholesterol content had been increased by 72-fold by feeding a high-cholesterol, high-fat diet. These studies suggest that the late endosomal/lysosomal content of unesterified cholesterol correlates with cell damage in NPC disease.     

Blood dolichol in lysosomal diseases.

Highly elevated serum total dolichol (free dolichol + dolichyl ester) concentrations have recently been found in two lysosomal storage diseases, aspartylglucosaminuria (AGU) and mannosidosis. The present study demonstrates that the increase of serum dolichol in AGU patients is caused by an increase of serum free dolichol. In 15 patients the mean serum level of free dolichol (227 +/- 16 ng/mL) was 1.9 times higher (p < 0.001) than that in healthy controls (120 +/- 6 ng/mL), while the amounts of dolichol fatty acid esters were similar in the patients and controls (110 +/- 9 vs. 118 +/- 6 ng/mL). In contrast, 10 patients with neuronal ceroid-lipofuscinosis (NCL) (three with infantile, four with juvenile, and three with variant late infantile NCL) had significantly (p < 0.01) lower mean serum levels of both free (79 +/- 5 ng/mL) and total (159 +/- 6 ng/mL) dolichol than age-adjusted healthy controls (free, 100 +/- 6 ng/mL; total, 206 +/- 14 ng/mL). Decreased blood dolichol has not been reported earlier for any other disease. We conclude that the increased serum free dolichol in AGU reflects disturbed lysosomal function and that the decreased free and esterified dolichols in NCLs speak against their presumed primary lysosomal nature.     

Increased hepatic dolichol and dolichyl phosphate-mediated glycosylation in rats fed cholesterol

The concentrations of dolichol and cholesterol in livers of rats maintained for 2 weeks on a diet enriched with cholesterol (1%) were significantly higher than those in animals on a normal diet. The incorporation of radioactive mevalonate into dolichol and into a dolichyl diphosphate oligosaccharide fraction by liver slices of the cholesterol-fed animals was increased over that of the control group. However, the incorporation of radioactive mevalonate into cholesterol was decreased, as was the incorporation of radioactive acetate into both dolichol and, more markedly, cholesterol. These results are consistent with cholesterol feeding causing partial inhibition of the cholesterol-biosynthetic pathway both at β-hydroxy-β-methylglutaryl coenzyme A reductase and at a step after farnesyl pyrophosphate formation, resulting in a greater flux of mevalonate to dolichol and an increase in pool sizes of precursors of β-hydroxy-β-methylglutaryl coenzyme A. Maximal activity of glycosyl transfer to dolichyl phosphate was greater in microsomal preparations from livers of cholesterol-fed animals compared with those of control animals. A corresponding higher degree of in vitro glycosylation of endogenous protein was also observed. It is concluded that the cholesterol-enriched diet caused an increase in the biosynthesis and concentration of dolichyl monophosphate which resulted in a higher level of N-glycosylation of protein. These effects were complicated by differences in the kinetics of glycosyl transfer and in its response to exogenous dolichyl monophosphate.     

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.     

Overexpression of sterol carrier protein-2 differentially alters hepatic cholesterol accumulation in cholesterol-fed mice

Although in vitro studies suggest a role for sterol carrier protein-2 (SCP-2) in cholesterol trafficking and metabolism, the physiological significance of these observations remains unclear. This issue was addressed by examining the response of mice overexpressing physiologically relevant levels of SCP-2 to a cholesterol-rich diet. While neither SCP-2 overexpression nor cholesterol-rich diet altered food consumption, increased weight gain, hepatic lipid, and bile acid accumulation were observed in wild-type mice fed the cholesterol-rich diet. SCP-2 overexpression further exacerbated hepatic lipid accumulation in cholesterol-fed females (cholesterol/cholesteryl esters) and males (cholesterol/cholesteryl esters and triacyglycerol). Primarily in female mice, hepatic cholesterol accumulation induced by SCP-2 overexpression was associated with increased levels of LDL-receptor, HDL-receptor scavenger receptor-B1 (SR-B1) (as well as PDZK1 and/or membrane-associated protein 17 kDa), SCP-2, liver fatty acid binding protein (L-FABP), and 3α-hydroxysteroid dehydrogenase, without alteration of other proteins involved in cholesterol uptake (caveolin), esterification (ACAT2), efflux (ATP binding cassette A-1 receptor, ABCG5/8, and apolipoprotein A1), or oxidation/transport of bile salts (cholesterol 7α-hydroxylase, sterol 27α-hydroxylase, Na⁺/taurocholate cotransporter, Oatp1a1, and Oatp1a4). The effects of SCP-2 overexpression and cholesterol-rich diet was downregulation of proteins involved in cholesterol transport (L-FABP and SR-B1), cholesterol synthesis (related to sterol regulatory element binding protein 2 and HMG-CoA reductase), and bile acid oxidation/transport (via Oapt1a1, Oatp1a4, and SCP-x). Levels of serum and hepatic bile acids were decreased in cholesterol-fed SCP-2 overexpression mice, especially in females, while the total bile acid pool was minimally affected. Taken together, these findings support an important role for SCP-2 in hepatic cholesterol homeostasis.     

Properties of a dolichol phosphokinase activity associated with rat liver microsomes

Rat liver microsomes show a capacity to synthesize [1-3H]dolichyl phosphate from [1-3H]-dolichol. Formation of [1-3H]dolichyl phosphate increased continuously over 15 min although the reaction rate was never completely linear. Product formation was directly proportional to microsomal protein concentration between 1.1 mg/mL and the highest concentration tested, 5.5 mg/mL. The reaction rate was linear with respect to the dolichol content of the assay mixture to a saturation point (120 microM). An apparent Km of 50 microM was established for dolichol. The normal phosphate donor for the reaction is CTP and not ATP. The optimum concentration of CTP was 10 mM, and an apparent Km of 4 mM was calculated for this nucleoside triphosphate. The reaction was totally dependent on divalent metal ion, magnesium being more effective than calcium. The optimum concentration of magnesium ion and CTP were the same (10 mM), suggesting that MgCTP2- is utilized as the normal enzyme substrate. Activity measured in the absence of Triton X-100 was only 5% of the activity observed at the optimum (0.5% w/v) detergent concentration. The measurable levels of dolichol phosphokinase could be doubled by the inclusion of 10-15 mM NaF as phosphatase inhibitor. Optimal enzymatic activity was obtained between pH 7.0 and pH 7.5 and could be inhibited by EDTA. The sulfhydryl reagent DTT was slightly stimulatory while the product of the reaction, dolichyl phosphate, was noninhibitory at the highest concentration tested (13.8 microM). The second reaction product (CDP) inhibits the enzymatic phosphorylation of dolichol.     

Separation of some neutral lipids by normal-phase high-performance liquid chromatography on a cyanopropyl column: Ubiquinone, dolichol, and cholesterol levels in sheep liver

The normal-phase high-performance liquid chromatographic separation of neutral lipids into molecular classes was carried out on a cyanopropyl (CN) column eluted with isopropanol in hexane. Cholesteryl, retinyl, and dolichyl esters, triglycerides and vitamin E, ubiquinone, dolichol, phytol, and cholesterol eluted as separate peaks with 0.05% isopropanol in hexane. Cholesterol, retinol, diglyceride, and monoglyceride eluted as separate peaks with 0.75% isopropanol in hexane. These separations could not be achieved on a silica gel column. The method was used to assay sheep liver ubiquinone, dolichol, and cholesterol levels, that were determined as 77, 108 and 1864 micrograms/g wet wt, respectively.     

The biosynthesis of hepatic cholesterol esters and triglycerides is impaired in mice with a disruption of the gene for stearoyl-CoA desaturase 1

Stearoyl-CoA desaturase (SCD) is a microsomal enzyme required for the biosynthesis of oleate and palmitoleate, which are the major monounsaturated fatty acids of membrane phospholipids, triglycerides, and cholesterol esters. Two well characterized isoforms of SCD, SCD1 and SCD2, exist in the mouse. Most mouse tissues express SCD1 and 2 with the exception of the liver, which expresses mainly the SCD1 isoform. We found that asebia mice homozygous for a natural mutation of the gene for SCD1 (SCD-/-) are deficient in hepatic cholesterol esters and triglycerides despite the presence of normal activities of acyl-CoA:cholesterol acyltransferase and glycerol phosphate acyltransferase, the enzymes responsible for cholesterol ester and triglyceride synthesis, respectively, in the liver of these mice. Feeding diets supplemented with triolein or tripalmitolein to the SCD-/- mice resulted in an increase in the levels of 16:1 and 18:1 in the liver but failed to restore the 18:1 and 16:1 levels of the cholesterol ester and triglycerides to the levels found in normal mice. The SCD-/- mouse had very low levels of triglycerides in the VLDL and LDL lipoprotein fractions compared with the normal animal. Transient transfection of an SCD1 expression vector into Chinese hamster ovary cells resulted in increased SCD activity and esterification of cholesterol to cholesterol esters. Taken together, our observations demonstrate that the oleoyl-CoA and palmitoleyl-CoA produced by SCD1 are necessary to synthesize enough cholesterol esters and triglycerides in the liver and suggest that regulation of SCD1 activity plays an important role in mechanisms of cellular cholesterol homeostasis.     

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.     

Liver fatty acid-binding protein gene ablation inhibits branched-chain fatty acid metabolism in cultured primary hepatocytes

Whereas the role of liver fatty acid-binding protein (L-FABP) in the uptake, transport, mitochondrial oxidation, and esterification of normal straight-chain fatty acids has been studied extensively, almost nothing is known regarding the function of L-FABP in peroxisomal oxidation and metabolism of branched-chain fatty acids. Therefore, phytanic acid (most common dietary branched-chain fatty acid) was chosen to address these issues in cultured primary hepatocytes isolated from livers of L-FABP gene-ablated (-/-) and wild type (+/+) mice. These studies provided three new insights: First, L-FABP gene ablation reduced maximal, but not initial, uptake of phytanic acid 3.2-fold. Initial uptake of phytanic acid uptake was unaltered apparently due to concomitant 5.3-, 1.6-, and 1.4-fold up-regulation of plasma membrane fatty acid transporter/translocase proteins (glutamic-oxaloacetic transaminase, fatty acid transport protein, and fatty acid translocase, respectively). Second, L-FABP gene ablation inhibited phytanic acid peroxisomal oxidation and microsomal esterification. These effects were consistent with reduced cytoplasmic fatty acid transport as evidenced by multiphoton fluorescence photobleaching recovery, where L-FABP gene ablation reduced the cytoplasmic, but not membrane, diffusional component of NBD-stearic acid movement 2-fold. Third, lipid analysis of the L-FABP gene-ablated hepatocytes revealed an altered fatty acid phenotype. Free fatty acid and triglyceride levels were decreased 1.9- and 1.6-fold, respectively. In summary, results with cultured primary hepatocytes isolated from L-FABP (+/+) and L-FABP (-/-) mice demonstrated for the first time a physiological role of L-FABP in the uptake and metabolism of branched-chain fatty acids.     

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.     

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.