M.J.A. van Wijland

Regulation of biliary lipid secretion by mdr2 P-glycoprotein in the mouse.

Disruption of the mdr2 gene in mice leads to a complete absence of phospholipid from bile (Smit, J. J. M., et al. 1993. Cell. 75:451-462). We have investigated the control of both mdr2 P-glycoprotein (Pgp) expression and bile salt secretion on biliary lipid secretion in the mouse. Lipid secretion was monitored at various bile salt output rates in wild-type mice (+/+), heterozygotes (+/-), and homozygotes (-/-) for mdr2 gene disruption. In (-/-) mice, phospholipid secretion was negligible at all bile salt output rates. In (+/-) mice, a curvilinear relation between bile salt and phospholipid secretion was observed similar to that in (+/+) mice; however, at all bile salt secretion rates phospholipid secretion was reduced compared to (+/+) mice, indicating that mdr2 Pgp exerts a strong control over secretion. Infusion of increasing amounts of taurocholate up to maximal secretory rate led to a decline in the phospholipid and cholesterol secretion in both (+/+) and (+/-) mice in accordance to what has been observed in other species. In contrast, in (-/-) mice cholesterol secretion increased under these conditions while phospholipid output remained extremely low. The increased cholesterol secretion may represent extraction of cholesterol from the canalicular plasma membrane by taurocholate micelles as opposed to the concomitant secretion of both phospholipid and cholesterol in the presence of a functional mdr2 Pgp. Increased bile flow in (-/-) mice could be attributed completely to an increase in the bile salt-independent fraction and may therefore be caused by the bile duct proliferation in these mice.

Direct demonstration that the deficient oxidation of very long chain fatty acids in X-linked adrenoleukodystrophy is due to an impaired ability of peroxisomes to activate very long chain fatty acids

A method was developed to prepare peroxisome-enriched fractions depleted of microsomes and mitochondria from cultured skin fibroblasts. The method consists of differential centrifugation of a postnuclear supernatant followed by density gradient centrifugation on a discontinuous Metrizamide gradient. The activity of hexacosanoyl-CoA synthetase was subsequently measured in postnuclear supernatants and peroxisome-enriched fractions prepared from cultured skin fibroblasts from control subjects and patients with X-linked adrenoleukodystrophy. Whereas the hexacosanoyl-CoA synthetase activity in postnuclear supernatants of X-linked adrenoleukodystrophy fibroblasts was only slightly decreased (77.8 +/- 4.4% of control (n = 15], enzyme activity was found to be much more markedly reduced in peroxisomal fractions isolated from the mutant fibroblasts (19.6 +/- 6.7% of control (n = 5]. This is a direct demonstration that the defect in X-linked adrenoleukodystrophy is at the level of a deficient ability of peroxisomes to activate very long chain fatty acids, as first suggested by Hashmi et al. [Hashmi, M., Stanley, W. and Singh, I. (1986) FEBS Lett. 86, 247-250].

Peroxisomal fatty acid beta-oxidation in relation to the accumulation of very long chain fatty acids in cultured skin fibroblasts from patients with Zellweger syndrome and other peroxisomal disorders.

The peroxisomal oxidation of the long chain fatty acid palmitate (C16:0) and the very long chain fatty acids lignocerate (C24:0) and cerotate (C26:0) was studied in freshly prepared homogenates of cultured skin fibroblasts from control individuals and patients with peroxisomal disorders. The peroxisomal oxidation of the fatty acids is almost completely dependent on the addition of ATP, coenzyme A (CoA), Mg2+ and NAD+. However, the dependency of the oxidation of palmitate on the concentration of the cofactors differs markedly from that of the oxidation of lignocerate and cerotate. The peroxisomal oxidation of all three fatty acid substrates is markedly deficient in fibroblasts from patients with the Zellweger syndrome, the neonatal form of adrenoleukodystrophy and the infantile form of Refsum disease, in accordance with the deficiency of peroxisomes in these patients. In fibroblasts from patients with X-linked adrenoleukodystrophy the peroxisomal oxidation of lignocerate and cerotate is impaired, but not that of palmitate. Competition experiments indicate that in fibroblasts, as in rat liver, distinct enzyme systems are responsible for the oxidation of palmitate on the one hand and lignocerate and cerotate on the other hand. Fractionation studies indicate that in rat liver activation of cerotate and lignocerate to cerotoyl-CoA and lignoceroyl-CoA, respectively, occurs in two subcellular fractions, the endoplasmic reticulum and the peroxisomes but not in the mitochondria. In homogenates of fibroblasts from patients lacking peroxisomes there is a small (25%) but significant deficiency of the ability to activate very long chain fatty acids. This deficient activity of very long chain fatty acyl-CoA synthetase is also observed in fibroblast homogenates from patients with X-linked adrenoleukodystrophy. We conclude that X-linked adrenoleukodystrophy is caused by a deficiency of peroxisomal very long chain fatty acyl-CoA synthetase.

X-linked adrenoleukodystrophy: defective peroxisomal oxidation of very long chain fatty acids but not of very long chain fatty acyl-CoA esters

We investigated the peroxisomal fatty acid beta-oxidation system in liver and cultured skin fibroblasts from patients with X-linked adrenoleukodystrophy known to accumulate very long chain fatty acids. In order to examine whether the deficient peroxisomal oxidation of very long chain fatty acids in these patients results from a deficiency in one of the peroxisomal beta-oxidation enzyme proteins (acyl-CoA oxidase, bifunctional protein with enoyl-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase activities and 3-oxoacyl-CoA thiolase) we carried out immunoblotting experiments using antibodies directed against the peroxisomal beta-oxidation enzyme proteins from rat liver. Furthermore, we studied the oxidation of palmitoyl-CoA and lignoceroyl-CoA in homogenates of fibroblasts from the patients. The results indicate that the peroxisomal beta-oxidation enzyme proteins are not only present immunologically but also functionally active which suggests that the defect in X-linked adrenoleukodystrophy is, indeed, as recently suggested by Hashmi and coworkers (FEBS Lett 1986;196:247-250) at the level of a deficient peroxisomal activation of very long chain fatty acids.

Peroxisomal very long-chain fatty acid β-oxidation in human skin fibroblasts: activity in Zellweger syndrome and other peroxisomal disorders

Since very long-chain fatty acids with a chain length of 24 carbons or more are known to accumulate in tissues and body fluids from patients with the cerebrohepato-renal (Zellweger) syndrome, infantile Refsum disease, neonatal adrenoleukodystrophy and X-linked adrenoleukodystrophy, we studied very long-chain fatty acid oxidation in cultured skin fibroblasts from these patients. In this paper, we report that in accordance with earlier results the first step in the beta-oxidation of the very long-chain fatty acid lignoceric acid (C24:0) primarily occurs in peroxisomes in control human skin fibroblasts. Furthermore, it was found that peroxisomal lignoceric acid beta-oxidation was strongly deficient in fibroblasts from patients with Zellweger syndrome, infantile Refsum disease, neonatal and X-linked adrenoleukodystrophy, which explains for the accumulation of very long-chain fatty acids in all four disease entities. In Zellweger syndrome, infantile Refsum disease and neonatal adrenoleukodystrophy the impairment in peroxisomal very long-chain fatty acid beta-oxidation is probably caused by a strong deficiency of all peroxisomal beta-oxidation enzyme proteins due to a deficiency of peroxisomes.

Studies on the peroxisomal oxidation of palmitate and lignocerate in rat liver

We have investigated the pathways involved in the peroxisomal oxidation of palmitate and lignocerate, measured as the cyanide-insensitive formation of acetyl units, in rat-liver homogenates. The peroxisomal beta-oxidation of both fatty acids is dependent on the presence of ATP, coenzyme A, NAD+ and Mg2+. However, there is a striking difference in the dependence of the rate of oxidation of the two substrates on the concentration of the individual cofactors, especially ATP. The peroxisomal beta-oxidation of lignocerate was inhibited to a progressively greater extent by increasing concentrations of palmitate and vice versa. Activation of lignoceric acid to lignoceroyl-CoA, however, was not inhibited by increasing concentrations of palmitate, and vice versa. It can be concluded that the peroxisomal palmitate and lignocerate beta-oxidation pathways differ in at least one enzymic reaction (the synthetase), but that the two pathways share at least one common step.

X-Linked Adrenoleukodystrophy: Identification of the Primary Defect at the Level of a Deficient Peroxisomal Very Long Chain Fatty Acyl-CoA Synthetase Using a Newly Developed Method for the Isolation of Peroxisomes from Skin Fibroblasts

Several types of adrenoleukodystrophy (ALD) differing in age of onset, mode of inheritance and clinical presentation have been described in the literature. The X-linked form of adrenoleukodystrophy, which is the most common, is characterized by demyelination, adrenal sufficiency and the accumulation of very long chain fatty acids, particularly hexacosanoic acid (C26:0), in tissues and body fluids (Moser et al., 1984). It is now generally accepted that the accumulation of very long chain fatty acids in X-linked ALD is caused by their impaired degradation via the peroxisomal β-oxidation system as first shown by Singh and co-workers in 1981 (see also Rizzo et al., 1984; Singh et al., 1984; Tsuji et al., 1985). Recent studies have shown that the deficient peroxisomal oxidation of very long chain fatty acids in X- linked ALD is not caused by a deficiency of one of the peroxisomal β-oxidation enzymes (acyl-CoA oxidase, bifunctional protein with enoyl-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase activities and 3-oxoacyl-CoA thiolase) since all three enzyme proteins were not only found to be immunologically present as shown by immunoblotting (Wanders et al., 1987) but also functionally active (Hashmi et al., 1986; Wanders et al., 1987). The latter finding led Hashmi and co-workers to suggest that the defect in X-linked ALD is at the level of a deficient activation of very long chain fatty acids to their CoA-esters. However, measurement of this activity in X-linked ALD fibroblasts using tetracosanoic acid (C24:0) and hexacosanoic acid (C26:0) as substrates revealed only a partial (about 25%) very long chain acyl-CoA synthetase deficiency (Wanders et al., 1987a).

Peroxisomes and peroxisomal functions in hyperpipecolic acidaemia

Hyperpipecolic acidaemia (McKusick 23940) has so far been reported in only four patients (Gatfield et al., 1968; Thomas et al., 1975; Burton et al., 1981). The hallmarks of this disorder are delayed development, hepatomegaly, hypotonia, retinopathy and progressive neurological deterioration with death occurring before 2–2½ years of age. Loading tests performed in three of the patients suggested a block in pipecolic acid degradation.

Peroxisomal fatty acid β-oxidation in human skin fibroblasts: X-linked adrenoleukodystrophy, a peroxisomal very long chain fatty acyl-CoA synthetase deficiency?

R. J. A. WANDERS l*, C. W. T. VAN ROERMtJND 1, M. J. A. VAN WIJLAND 1, J. HEIKOOP 1, A. VAN DEN PUT 1, P. BENTLAGE 1, E. MEIJBOOM 1, J. M. TAGER 2, A. W. SCHRAM 2, H. VAN DEN BOSCH 3 and R. B. H. SCHUTGENS 1 aDepartment of Pediatrics, University of Amsterdam, Meibergdreef 9, 1105 A Z Amsterdam, The Netherlands; 2Laboratory of Biochemistry, University of Amsterdam, Meibergdreef 15, 1105 A Z Amsterdam, The Netherlands; ~Laboratory of Biochemistry, State University Utrecht, Padualaan 8, 3584 CH Utrecht, The Netherlands

Identification of the Enzymic Defect in X-Linked Adrenoleukodystrophy: Oxidation of Very Long Chain Fatty Acids is Deficient Due to an Impaired Ability of Peroxisomes to Activate Very Long Chain Fatty Acids

Mitochondria were long thought to be the sole site of fatty acid β-oxidation. Following the discovery by Cooper and Beevers1 that castor bean endosperm glyoxysomes, organelles closely related to peroxisomes, contain a fatty acid β-oxidation system, peroxisomes from rat liver2 and various other mammalian tissues were subsequently found to catalyze fatty acyl-CoA β-oxidation as well (reviewed in Refs. 3-6). Like mitochondrial fatty acid β-oxidation, peroxisomal fatty acid β-oxidation proceeds via successive steps of dehydrogenation, hydration, dehydrogenation and thiolytic cleavage. These reactions are catalyzed by the specific peroxisomal β-oxidation enzyme proteins acyl-CoA oxidase (EC 1.3.99.3), the bifunctional protein with enoyl-CoA hydratase (EC 4.2.1.17) and 3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35) activities and 3-oxoacyl-CoA thiolase (EC 2.3.1.16).7 Despite the apparent similarities in architecture between the β-oxidation systems in peroxisomes and mitochondria, there are a number of important differences between the two systems with regard to the properties of the individual enzymes, cofactor requirements, coupling to energy production etc. Moreover, the peroxisomal and mitochondrial β-oxidation systems have different substrate specificities. Studies by Moser and coworkers for instance have shown that very long chain fatty acids are primarily oxidized in peroxisomes, at least in rat liver.8 That this is also true in man is indicated by the finding of elevated very long chain fatty acid levels in tissues and body fluids from patients suffering from the cerebro-hepato-renal syndrome of Zellweger in which morphologically distinguishable peroxisomes are known to be absent.9 Recent immunoblotting studies have shown that the three peroxisomal β-oxidation enzyme proteins are strongly deficient in liver from Zellweger patients,10-12 which explains the impairment in peroxisomal very long chain fatty acid β-oxidation8,13-16 leading to elevated very long chain fatty acid levels in these patients.