Rhizomelic chondrodysplasia punctata: biochemical studies of peroxisomes isolated from cultured skin fibroblasts.

Peroxisomes isolated from cultured skin fibroblasts of two patients with rhizomelic chondrodysplasia punctata (RCDP) and two controls were compared for biochemical studies. These experiments provided the following results: (1) peroxisomes isolated from RCDP-cultured skin fibroblasts had the same density (1.175 g/ml) as control peroxisomes; (2) dihydroxyacetone phosphate acyltransferase activity, the first enzyme in the synthesis of plasmalogens, was deficient (0.5% of control) in RCDP peroxisomes and this activity was not observed in any other region of the gradient; (3) the rate of activation (lignoceroyl-CoA ligase) and oxidation of lignoceric acid was normal in RCDP peroxisomes; and (4) peroxisomes from RCDP contained 3-ketoacyl-CoA thiolase in the unprocessed form (44-kDa protein), whereas control peroxisomes had both processed (41-kDa protein) and unprocessed forms of 3-ketoacyl-CoA thiolase. The presence of both processed and unprocessed 3-ketoacyl-CoA thiolase in control peroxisomes and the unprocessed form in RCDP peroxisomes suggests that processing of 3-ketoacyl-CoA thiolase takes place in peroxisomes. Although the specific activity and percentage of activity of 3-ketoacyl-CoA thiolase in RCDP peroxisomes was only 22-26% of control, the normal oxidation of lignoceric acid in RCDP peroxisomes indicates that unprocessed 3-ketoacyl-CoA thiolase is active. The remaining peroxisomal 3-ketoacyl-CoA thiolase activity in RCDP was observed in a protein fraction (peroxisome ghosts) lighter than peroxisomes. The normal oxidation of fatty acids in peroxisomes and the absence of such activity in peroxisome ghosts (d = 1.12 g/ml) containing peroxisomal proteins in RCDP suggest that RCDP has only one population of functional peroxisomes (d = 1.175 g/ml).     

The effect of diabetes mellitus on aortic prostanoid synthesis and serum cholesterol levels in the rat fed a high cholesterol diet

The effect of diabetes mellitus on serum cholesterol and aortic microsomal prostanoid synthesis was studied in cholesterol fed male Lewis rats. Normal, diabetic and diabetic rats treated with pancreatic islets were divided into three diet subgroups, control diet, control +2% cholesterol for 8 weeks and control +2% cholesterol diet for 16 weeks. Serum glucose levels were elevated three-fold in the diabetic group compared to normal. Treatment with islets restored serum glucose to normal levels in diabetic rats. The 2% cholesterol diet did not significantly alter serum glucose levels in any of the groups. Body weights in the diabetic group were significantly lower than normal or diabetic rats treated with islets. Feeding 2% cholesterol for 16 weeks significantly increased weight in normal and islet treated diabetic rats but not in the diabetic group. Aortic microsomal prostanoid synthesis was similar in all experimental groups with 6-keto-PGF1 alpha (PGI2 metabolite) being the major product synthesized in all groups. Aortic microsomal prostanoid levels were not altered by the 2% cholesterol diet. Serum cholesterol levels increased 14-fold in the diabetic group which returned to the normal level in the diabetic animals treated with islets. These data show that diabetes does not alter aortic microsomal prostanoid levels in the rat. However, diabetes significantly increased serum cholesterol levels which were reversed by islet transplantation.     

Peroxisomal protein targeting and identification of peroxisomal targeting signals in cholesterol biosynthetic enzymes

At least three different subcellular compartments, including peroxisomes, are involved in cholesterol synthesis. Recently, it has been demonstrated that peroxisomes contain a number of enzymes involved in cholesterol biogenesis that previously were considered to be cytosolic or located in the endoplasmic reticulum. Peroxisomes have been shown to contain acetoacetyl-CoA thiolase, HMG-CoA synthase, HMG-CoA reductase, mevalonate kinase, phosphomevalonate kinase, phosphomevalonate decarboxylase, isopentenyl diphosphate isomerase and FPP synthase. Moreover, the activities of these enzymes are also significantly decreased in liver tissue and fibroblast cells obtained from patients with peroxisomal deficiency diseases. In addition, the cholesterol biosynthetic capacity is severely impaired in cultured skin fibroblasts obtained from patients with peroxisomal deficiency diseases. These findings support the proposal that peroxisomes play an essential role in isoprenoid biosynthesis. This paper presents a review of peroxisomal protein targeting and of recent studies demonstrating the localization of cholesterol biosynthetic enzymes in peroxisomes and the identification of peroxisomal targeting signals in these proteins.     

Cultured fibroblasts from patients with Niemann-Pick disease type C and type D exhibit distinct defects in cholesterol esterification.

The Niemann-Pick group of diseases can be broadly classified into two types based on clinical and biochemical characteristics. Type I is characterized by a primary deficiency of lysosomal sphingomyelinase while Type II may have a defect in the regulation of intracellular cholesterol metabolism. We have studied cholesterol esterification in cultured fibroblasts from patients with two phenotypes of Type II disease: an Acadian population of southwestern Nova Scotia (Canada) with a form of the disease known as Niemann-Pick type D (NPD) and a group of panethnic origin with Niemann-Pick type C (NPC). Addition of whole serum to normal fibroblasts grown initially in lipoprotein-deficient serum caused a rapid (within 6 h) increase in cholesterol esterification, reaching maximum values at around 24 h, while NPC fibroblasts showed little increase (less than 10% of normal). In contrast, cholesterol esterification in NPD fibroblasts increased slowly during the first 6-12 h and reached 50% of normal values by 24 h. 25-Hydroxycholesterol, a non-lipoprotein stimulator of cholesterol esterification, caused a similar stimulation of cholesterol esterification in NPC, NPD and normal cells. This was inhibited by addition of serum in mutant but not in normal cells. Within 24 h of serum addition, free cholesterol accumulated in all cell types with NPC greater than NPD greater than normal. These observations indicate that (a) regulation of cholesterol esterification in response to serum lipoproteins (but not 25-hydroxycholesterol) is abnormal in both NPC and NPD fibroblasts, and (b) the biochemical phenotypes of fibroblasts from NPC and NPD patients are distinct.     

Peroxisomal cholesterol biosynthesis and Smith-Lemli-Opitz syndrome

Smith-Lemli-Opitz syndrome (SLOS), caused by 7-dehydrocholesterol-reductase (DHCR7) deficiency, shows variable severity independent of DHCR7 genotype. To test whether peroxisomes are involved in alternative cholesterol synthesis, we used [1-(14)C]C24:0 for peroxisomal beta-oxidation to generate [1-(14)C]acetyl-CoA as cholesterol precursor inside peroxisomes. The HMG-CoA reductase inhibitor lovastatin suppressed cholesterol synthesis from [2-(14)C]acetate and [1-(14)C]C8:0 but not from [1-(14)C]C24:0, implicating a peroxisomal, lovastatin-resistant HMG-CoA reductase. In SLOS fibroblasts lacking DHCR7 activity, no cholesterol was formed from [1-(14)C]C24:0-derived [1-(14)C]acetyl-CoA, indicating that the alternative peroxisomal pathway also requires this enzyme. Our results implicate peroxisomes in cholesterol biosynthesis but provide no link to phenotypic variation in SLOS.     

Ultrastructural and cytochemical demonstration of peroxisomes in cultured fibroblasts from patients with peroxisomal deficiency disorders

The oxidation of very long chain fatty acids and synthesis of ether glycerolipids (plasmalogens) occurs mainly in peroxisomes. Zellweger's cerebrohepatorenal syndrome (CHRS) is a rare, inherited metabolic disease characterized by an apparent absence of peroxisomes, an accumulation of very long chain fatty acids, and a decrease of plasmalogens in tissues and cultured fibroblasts from these patients. As peroxisomes are ubiquitous in mammalian cells, we examined normal and CHRS-cultured fibroblasts for their presence, using an electron microscopic histochemical procedure for the subcellular localization of catalase, a peroxisomal marker enzyme. Small (0.08-0.20 micron) round or slightly oval peroxisomes were seen in both normal and CHRS fibroblasts. The number of peroxisomes was analyzed morphometrically and found to be significantly reduced in all CHRS cell lines. These results are discussed in relation to the underlying defect in peroxisomal function and biogenesis in this disease.     

Rat liver peroxisomes catalyze the initial step in cholesterol synthesis. The condensation of acetyl-CoA units into acetoacetyl-CoA

In the last few years, it has been demonstrated by this group and others that rat liver peroxisomes participate in cholesterol synthesis. It has been shown that the key regulatory enzyme of isoprenoid biosynthesis, 3-hydroxy-3-methylglutaryl coenzyme A reductase, is present in liver cells not only in the endoplasmic reticulum but also within peroxisomes. It has been also demonstrated that rat liver peroxisomes in the presence of cytosolic proteins in vitro are able to convert [14C]mevalonic acid to [14C]cholesterol. In addition, a recent study demonstrated that the largest cellular concentration of sterol carrier protein-2 is inside peroxisomes. It is of interest, therefore, to inquire whether other proteins known to be involved in cholesterol biogenesis are also present in peroxisomes. In this study we investigated the first step in cholesterol synthesis, the condensation of two acetyl-CoA units to acetoacetyl-CoA. It was demonstrated that peroxisomal thiolase, purified by DEAE-phosphocellulose chromatography from gemfibrozil-treated rats, is active not only toward acetoacetyl-CoA and 3-ketoacyl-CoA, consistent with literature reports, but is also capable of converting acetyl-CoA units to acetoacetyl-CoA. This is the first demonstration of condensation activity in rat liver peroxisomes.     

Isoprenoid biosynthesis is not compromised in a Zellweger syndrome mouse model.

Because several studies indicated that peroxisomes are important for the biosynthesis of isoprenoids, we wanted to investigate whether a reduced availability of isoprenoids could be one of the pathogenic factors contributing to the severe phenotype of the Pex5(-/-) mouse, a model for Zellweger syndrome. Total cholesterol was determined in plasma, brain and liver of newborn mice. In none of these tissues a significant difference was observed between Pex5(-/-) and wild type or heterozygous mice. The hepatic ubiquinone content was found to be even higher in Pex5(-/-) mice as compared to wild type or heterozygous littermates. To investigate whether the Pex5(-/-) fetuses are able to synthesise their own isoprenoids, fibroblasts derived from these mice were incubated with radiolabeled mevalonolactone as a substrate for isoprenoid synthesis. No significant difference was observed between the cholesterol production rates of Pex5(-/-) and normal fibroblasts. Our results show that there is no deficiency of isoprenoids in newborn Pex5(-/-) mice, excluding the possibility that a lack of these compounds is a determinant factor in the development of the disease state before birth.     

Niemann-Pick type II fibroblasts exhibit impaired cholesterol esterification in response to sphingomyelin hydrolysis.

Fibroblasts from patients with Niemann-Pick Type II disease, including the panethnic type C (NPC) and Nova Scotia Acadian type D (NPD) forms, exhibit reduced or delayed stimulation of cholesterol esterification by low density lipoprotein (LDL). Based on recent evidence that cholesterol esterification can also be stimulated by cell surface sphingomyelin hydrolysis, we have compared the response of normal, NPC and NPD fibroblasts to treatment with exogenous sphingomyelinase (SMase). Staphylococcus aureus SMase (greater than 0.05 U/ml) hydrolyzed over 90% of endogenous sphingomyelin within 1 h and increased incorporation of [3H]oleic acid into cholesterol-[3H]oleate after an initial lag in all three cell types. However, normal levels of cholesterol esterification were not observed for NP Type II fibroblasts: four NPD cell lines exhibited an average of 32% of normal response while cholesterol esterification was only 20% in two well-characterized NPC lines. A third NPC line exhibited normal response to SMase despite greater than 90% impairment of LDL-stimulated cholesterol esterification. Incubation of fibroblasts with LDL followed by SMase produced a synergistic response, particularly in NPC cells where there was little response to either treatment alone. Chloroquine abolished LDL-stimulated cholesterol esterification in normal fibroblasts but had no effect on the response to SMase, indicating that lysosomal enzymes may not be involved in SMase-mediated cholesterol esterification. These results suggest that intracellular processing of cholesterol derived from either LDL or release from the plasma membrane (by sphingomyelin hydrolysis) is affected in Niemann-Pick Type II cells and that these pathways can complement one another in the stimulation of cholesterol esterification.     

A comparative study of straight chain and branched chain fatty acid oxidation in skin fibroblasts from patients with peroxisomal disorders.

The beta-oxidation of stearic acid and of alpha- and gamma-methyl isoprenoid-derived fatty acids (pristanic and tetramethylheptadecanoic acids, respectively) was investigated in normal skin fibroblasts and in fibroblasts from patients with inherited defects in peroxisomal biogenesis. Stearic acid beta-oxidation by normal fibroblast homogenates was several-fold greater compared to the oxidation of the two branched chain fatty acids. The effect of phosphatidylcholine, alpha-cyclodextrin, and bovine serum albumin on the three activities suggests that different enzymes are involved in the beta-oxidation of straight chain and branched chain fatty acids. Homogenates of fibroblasts from patients with a deficiency in peroxisomes (Zellweger syndrome and infantile Refsum's disease) showed a normal ability to beta-oxidize stearic acid, but the oxidation of pristanic and tetramethylheptadecanoic acid was decreased. Concomitantly, 14CO2 production from the branched chain fatty acids by Zellweger fibroblasts in culture (but not from stearic acid) was greatly diminished. The Zellweger fibroblasts also showed a marked reduction in the amount of water-soluble metabolites from the radiolabeled branched chain fatty acids that are released into the culture medium. The data presented indicate that the oxidation of alpha- and gamma-methyl isoprenoid-derived fatty acids takes place largely in peroxisomes in human skin fibroblasts.     

In vitro regulation of cholesterol metabolism by low density lipoproteins in skin fibroblasts from hypo-and hyperresponding squirrel monkeys.

When squirrel monkeys (Saimiri sciureus) are fed diets containing cholesterol, some individuals (hyperresponders) become hypercholesterolemic, while others (hyporesponders) are able to maintain nearly normal plasma cholesterol concentrations. Skin fibroblasts were grown from three hyperresponder and threehyporesponder squirrel monkeys, previously characterized on the basis of their plasma cholesterol response to two cholesterol-containing diets and the pheno-type of their parents. The rates of cholesterol synthesis and esterification were determined in the cultured fibroblasts incubated with low density lipoproteins isolated from normocholesterolemic squirrel monkeys or hypercholesterolemic rhesus monkeys. Both lipoprotein preparations influenced the metabolic parameters measured in a similar manner in cells from both hypo- and hyperresponder animals. Exposure of skin fibroblasts to low density lipoproteins resultd in a stimulation of cholesterol esterification and a suppression of cholesterol synthesis in cells from both hypo- and hyperresponder animals. When incubated with increasing concentrations of low density lipoprotein cholesterol, up to 50 microgram/ml, fibroblasts from both hypo-and hyperresponding animals responded with a similar maximum percentage suppression of sterol synthesis. Thus, hyperresponsiveness to dietary cholesterol in squirrel monkeys, although a heritable characteristic, is not associated with an inability of low density lipoprotein to suppress cholesterol synthesis or stimulate cholesterol esterification as occurs in familial hypercholesterolemia in man.     

Niemann-Pick Type II fibroblasts exhibit impaired cholesterol esterification in response to shingomyelin hydrolysis

Fibroblasts from patients with Niemann-Pick Type II disease, including the panethnic type C (NPC) and Nova Scotia Acadian type D (NPD) forms, exhibit reduced or delayed stimulation of cholesterol esterification by low density lipoprotein (LDL). Based on recent evidence that cholesterol esterification can also be stimulated by cell surface sphingomyelin hydrolysis, we have compared the response of normal, NPC and NPD fibroblasts to treatment with exogenous sphingomyelinase (SMase). Staphylococcus aureus SMase (> 0.05 U/ml) hydrolyzed over 90% of endogenous sphingomyelin within 1 h and increased incorporation of [³H]oleic acid into cholesterol-[³H]oleate after an initial lag in all three cell types. However, normal levels of cholesterol esterification were not observed for NP Type II fibroblasts: four NPD cell lines exhibited an average of 32% of normal response while cholesterol esterification was only 20% in two well-characterized NPC lines. A third NPC line exhibited normal response to SMase despite greater than 90% impairment of LDL-stimuated cholesterol esterification. Incubation of fibroblasts with LDL followed by SMase produced a synergistic response, particularly in NPC cells where there was little response to either treatment alone. Chloroquinone abolished LDL-stimulated cholesterol esterification in normal fibroblasts but had no effect on the response to SMase, indicating that lysosomal enzymes may not be involved in SMase-mediated cholesterol esterification. These results suggest that intracellular processing of cholesterol derived from either LDL or release from the plasma membrane (by sphingomyelin hydrolysis) is affected in Niemann-Pick Type II cells and that these pathways can complement one another in the stimulation of cholesterol esterification.     

Molecular mechanism of detectable catalase-containing particles, peroxisomes, in fibroblasts from a PEX2-defective patient

Patients with peroxisome biogenesis disorders (PBD) can be identified by detection of peroxisomes in their fibroblasts, by means of immunocytochemical staining using an anti-catalase antibody. We report here data on three PBD patients with newly identified mutations (del550C and del642G) in the PEX2 gene which encodes a 35-kDa peroxisomal membrane protein containing two membrane-spanning and a C-terminal cysteine-rich region. Some of the fibroblasts from the patient with the del642G mutation contained numerous catalase-containing particles, whereas no fibroblasts containing such particles were found in the patient with the del550C mutation. We confirmed that the del642G mutation caused a partial defect in peroxisome synthesis and import by expression of the mutated PEX2 into PEX2-defective CHO mutant cells. We propose that the two putative membrane-spanning segments in Pex2p are important domains for peroxisome assembly and import and that a defect in one of these domains severely affects PBD patients. Furthermore, a defect in the C-terminal portion of Pex2p exposed to the cytosol containing a RING finger motif caused the mild phenotype, residual enzyme activities, and mosaic detectable peroxisomes in fibroblasts from the patient.     

Lipid metabolism in peroxisomes in relation to human disease

Peroxisomes were long believed to play only a minor role in cellular metabolism but it is now clear that they catalyze a number of important functions. The importance of peroxisomes in humans is stressed by the existence of a group of genetic diseases in man in which one or more peroxisomal functions are impaired. Most of the functions known to take place in peroxisomes have to do with lipids. Indeed, peroxisomes are capable of 1, fatty acid β-oxidation 2, fatty acid α-oxidation 3, synthesis of cholesterol and other isoprenoids 4, ether-phospholipid synthesis and 5, biosynthesis of polyunsaturated fatty acids. In Chapter 2–6 we will discuss the functional organization and enzymology of these pathways in detail. Furthermore, attentin is paid to the permeability properties of peroxisomes with special emphasis on recent studies which suggest that peroxisomes are closed structures containing specific membrane proteins for trransport of metabolites. Finally, the disorders of peroxisomal lipid metabolism will be discussed.     

Localization of the pre-squalene segment of the isoprenoid biosynthetic pathway in mammalian peroxisomes

Previous studies have indicated that the early steps in the isoprenoid/cholesterol biosynthetic pathway occur in peroxisomes. However, the role of peroxisomes in cholesterol biosynthesis has recently been questioned in several reports concluding that three of the peroxisomal cholesterol biosynthetic enzymes, namely mevalonate kinase, phosphomevalonate kinase, and mevalonate diphosphate decarboxylase, do not localize to peroxisomes in human cells even though they contain consensus peroxisomal targeting signals. We re-investigated the subcellular localization of the cholesterol biosynthetic enzymes of the pre-squalene segment in human cells by using new stable isotopic techniques and data computations with isotopomer spectral analysis, in combination with immunofluorescence and cell permeabilization techniques. Our present findings clearly show and confirm previous studies that the pre-squalene segment of the cholesterol biosynthetic pathway is localized to peroxisomes. In addition, our data are consistent with the hypothesis that acetyl-CoA derived from peroxisomal β-oxidation of very long-chain fatty acids and medium-chain dicarboxylic acids is preferentially channeled to cholesterol synthesis inside the peroxisomes without mixing with the cytosolic acetyl-CoA pool.     

Peroxisomes and intracellular cholesterol trafficking in adult rat Leydig cells following Luteinizing hormone stimulation

The present study was designed to explore the intracellular cholesterol trafficking in Leydig cells of adult rats following Luteinizing hormone (LH) injection. Histochemical techniques were used to demonstrate distribution of free cholesterol in Leydig cells of control and LH-injected rats. Two groups of sexually mature male Sprague Dawley rats (n=4/group) were used. Fifteen min following an injection of 200 microl of either saline (control) or luteinizing hormone (LH, 500 microg in saline) testes of rats were fixed by whole body perfusion using 0.5% glutaraldehyde and 4% paraformaldehyde in 0.1 M cacodylate buffer for 20 min. Fixed testes were cut into 3 mm3 and kept immersed in the fixative for further 15 min. Tissue cubes were then incubated at 37 degrees C in a medium containing cholesterol oxidase, 3,3'-diaminobenzidine tetrahydrochloride, horseradish peroxidase and dimethyl sulfoxide to histochemically localize free cholesterol in Leydig cells and processed for electron microscopy. Thin sections of these tissues were stained with aqueous uranyl acetate and lead citrate and examined with a Philips 201C electron microscope. In Leydig cells of control rats, free cholesterol was detected primarily in lipid droplets and plasma membrane. In the majority of Leydig cells, peroxisomes were unstained for free cholesterol, but occasionally few stained ones were present. Staining was not detected in mitochondria and smooth endoplasmic reticulum (SER) in Leydig cells of control rats. In LH-injected rats, lipid droplets, many peroxisomes, inner and outer mitochondrial membranes and some cisternae of SER in Leydig cells showed staining for free cholesterol. Fusion of Leydig cell peroxisomes with lipid droplets and mitochondria was also observed in the LH treated rats. These findings suggested that peroxisomes in adult rat Leydig cells participate in the intracellular cholesterol trafficking and delivery into mitochondria during LH stimulated steroidogenesis. Lipid droplets are used as one source for cholesterol for this process.     

Concentration and composition of serum lipoproteins during a low-fat diet at two levels of polyunsaturated fat

A 12-week dietary intervention was carried out among 40 families from North Karelia, a county in Finland with an exceptionally high rate of coronary heart disease and high serum cholesterol values. The proportion of dietary energy derived from fat was reduced during the 12-week intervention period from about 39% to 23% in all families. The families were randomly allocated into two groups. Twenty families consumed a diet with a polyunsaturated to saturated fat (P/S) ratio of 0.9 (group I), while the other 20 families had a diet with a P/S ratio of 0.4 (group II). Total serum cholesterol decreased by 16% and 9% in men of groups I and II, respectively, and by 16% in women of both groups. These changes were due to a decrease in both low density lipoprotein (LDL) and high density lipoprotein (HDL) cholesterol. LDL cholesterol and phospholipid reached minimum values after 6 weeks on both intervention diets, but LDL protein responded more slowly. Thus, after 6 weeks LDL had an altered composition containing less cholesterol and phospholipids and more protein and triglycerides than during the baseline diet. During the intervention, the linoleic acid content in the serum cholesteryl ester fraction increased, and the magnitude of this change correlated negatively with the changes in total and LDL cholesterol. The decrease in HDL cholesterol during the two intervention diets was due to a fall in the HDL2 cholesterol (29% and 24% in men, and 26% and 25% in women in groups I and II, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)     

Mutation in PEX16 is causal in the peroxisome-deficient Zellweger syndrome of complementation group D.

Peroxisome-biogenesis disorders (PBDs), including Zellweger syndrome (ZS), are autosomal recessive diseases caused by a deficiency in peroxisome assembly as well as by a malfunction of peroxisomes, among which>10 genotypes have been identified. We have isolated a human PEX16 cDNA (HsPEX16) by performing an expressed-sequence-tag homology search on a human DNA database, by using yeast PEX16 from Yarrowia lipolytica and then screening the human liver cDNA library. This cDNA encodes a peroxisomal protein (a peroxin Pex16p) made up of 336 amino acids. Among 13 peroxisome-deficiency complementation groups (CGs), HsPEX16 expression morphologically and biochemically restored peroxisome biogenesis only in fibroblasts from a CG-D patient with ZS in Japan (the same group as CG-IX in the United States). Pex16p was localized to peroxisomes through expression study of epitope-tagged Pex16p. One patient (PBDD-01) possessed a homozygous, inactivating nonsense mutation, C-->T at position 526 in a codon (CGA) for 176Arg, that resulted in a termination codon (TGA). This implies that the C-terminal half is required for the biological function of Pex16p. PBDD-01-derived PEX16 cDNA was defective in peroxisome-restoring activity when expressed in the patient's fibroblasts. These results demonstrate that mutation in PEX16 is the genetic cause of CG-D PBDs.     

The cholesterol storage disorder of the mutant BALB/c mouse. A primary genetic lesion closely linked to defective esterification of exogenously derived cholesterol and its relationship to human type C Niemann-Pick disease

An inborn murine cholesterol storage disorder exists which is characterized by a lesion in intracellular cholesterol esterification not accounted for by any discernible abnormality in acyl-CoA: cholesterol acyltransferase (Pentchev, P.G., Boothe, A.D., Kruth, H.S., Weintroub, H., Stivers, J., and Brady, R.O. (1984) J. Biol. Chem. 259, 5784-5791). Current studies have shown that the level of esterification of nonlipoprotein-derived [3H]cholesterol in cultured fibroblasts from heterozygous mutant mice was intermediary between the level found in normal fibroblasts and the deficient level found in fibroblasts from homozygous mutant mice. Homozygous-affected fibroblasts took up and converted [3H]desmosterol to [3H]cholesterol at a normal rate indicating that the murine mutation does not compromise the transport of exogenous sterol to microsomes. In contrast to the defect in esterification of exogenously derived cholesterol, synthesis of cholesteryl ester from [3H]mevalonic acid and [3H]squalene was normal in affected fibroblasts as was the stimulation of cholesteryl ester synthesis from endogenous cholesterol induced by 25-hydroxycholesterol. In surveying a number of mutant cell lines from human metabolic disorders with phenotypic manifestations similar in part to the mutant cholesterol storage mouse, Niemann-Pick C fibroblasts displayed a similar defect in esterification of exogenously derived cholesterol.