Maurice Bugaut

Phenylbutyrate up-regulates the adrenoleukodystrophy-related gene as a nonclassical peroxisome proliferator

X-linked adrenoleukodystrophy (X-ALD) is a demyelinating disease due to mutations in the ABCD1 (ALD) gene, encoding a peroxisomal ATP-binding cassette transporter (ALDP). Overexpression of adrenoleukodystrophy-related protein, an ALDP homologue encoded by the ABCD2 (adrenoleukodystrophy-related) gene, can compensate for ALDP deficiency. 4-Phenylbutyrate (PBA) has been shown to induce both ABCD2 expression and peroxisome proliferation in human fibroblasts. We show that peroxisome proliferation with unusual shapes and clusters occurred in liver of PBA-treated rodents in a PPARα-independent way. PBA activated Abcd2 in cultured glial cells, making PBA a candidate drug for therapy of X-ALD. The Abcd2 induction observed was partially PPARα independent in hepatocytes and totally independent in fibroblasts. We demonstrate that a GC box and a CCAAT box of the Abcd2 promoter are the key elements of the PBA-dependent Abcd2 induction, histone deacetylase (HDAC)1 being recruited by the GC box. Thus, PBA is a nonclassical peroxisome proliferator inducing pleiotropic effects, including effects at the peroxisomal level mainly through HDAC inhibition.

Determination of bovine butterfat triacylglycerols by reversed-phase liquid chromatography and gas chromatography.

Triacylglycerols (TGs) from a sample of summer butterfat (bovine milk) were analysed and fractionated by reversed-phase liquid chromatography (RPLC). Fatty acid and TG composition of eac of the 47 RPLC fractions ranging from 0.1 to 6.9% were determined by capillary gas chromatography. The data were used together to determine the quantitative composition of the molecular species of TGs. A large number of TG species, accounting for 80% of the total, could be unequivocally identified and individually determined. The combination of the chromatographic methods used proved to be a powerful and accurate approach for the determination of molecular species of TGs in a complex fat, but also a difficult and time-consuming task.

Expression of peroxisome proliferator-activated receptors alpha and gamma in differentiating human colon carcinoma Caco-2 cells

The expression of peroxisome proliferator‐activated receptors α (PPARα) and γ (PPARγ) was studied in the human adenocarcinoma Caco‐2 cells induced to differentiate by long term culture (15 days). The differentiation of Caco‐2 cells was attested by increases in the activities of sucrase‐isomaltase and alkaline phosphatase (two brush border enzymes), fatty acyl‐CoA oxidase (AOX) and catalase (two peroxisomal enzymes), by an elevation in the protein levels of villin (a brush border molecular marker), AOX, peroxisomal bifunctional enzyme (PBE), catalase and peroxisomal membrane protein of 70 kDa (PMP70), and by the appearance of peroxisomes. The expression of PPARα and PPARγ was investigated by Western blotting, immunocytochemistry, Northern blotting and S1 nuclease protection assay during the differentiation of Caco‐2 cells. The protein levels of PPARα, PPARγ, and PPARγ2 increased gradually during the time‐course of Caco‐2 cell differentiation. Immunocytochemistry revealed that PPARα and γ were localized in cell nuclei. The PPARγ1 protein was encoded by PPARγ3 mRNA because no signal was obtained for PPARγ1 mRNA using a specific probe in S1 nuclease protection assay. The amount of PPARγ3 mRNA increased concomitantly to the resulting PPARγ1 protein. On the other hand, the mRNA of PPARα and PPARγ2 were not significantly changed, suggesting that the increase in their respective protein was due to an elevation of the translational rate. The role played by the PPAR subtypes in Caco‐2 cell differentiation is discussed.

Proteins and enzymes of the peroxisomal membrane in mammals

Summary— Proteins of the peroxisomal membrane can be schematically divided into two groups, one being made up of more or less characterized proteins with generally unknown functions and the other consisting of enzyme activities of which the corresponding proteins have not been characterized. In the present report, these proteins and enzymes are described with the addition of unpublished results regarding their induction by peroxisome proliferators at the post‐transcriptional level. Integral membrane proteins (IMPs) can be isolated using an alkaline solution of sodium carbonate. A dozen of preponderant IMPs can be seen on sodium dodecyl sulfate polyacrylamide gel electrophoresis, and the major band corresponds to a 70 kDa IMP, of which the corresponding rat cDNA is known. Some IMPs have been characterized by immunoblot analysis. Recently, a cDNA has been cloned for a peroxisome assembly factor (35 kDa IMP). Functions have also been proposed for some IMPs but are not yet firmly settled. Some IMPs (450/520, 70 and 26 kDa) are strongly induced by peroxisome proliferators. Our results extend to cipro‐ and fenofibrate the observation that the 70 kDa IMP mRNA level is strongly increased in di(2‐ethylhexyl)phtalate‐treated rats. All the enzyme activities associated with the peroxisomal membrane are involved in lipid metabolism: activation of substrates (fatty acids), ether lipid biosynthesis, and formation of precursors (fatty alcohols). It is believed that the same long‐chain acyl‐CoA synthetase occurs in the peroxisome as well as in the outer mitochondrial membrane and the endoplasmic reticulum. However, two highly homologous but different cDNAs encoding rat liver and brain long‐chain acyl‐CoA synthetases have been isolated recently. Evidence has been accumulated for a distinct synthetase that specifically activates very‐long chain fatty acids. The first two steps of ether lipid biosynthesis requiere dihydroxyacetone‐phosphate (DHAP) acyltransferase and alkyl‐DHAP synthetase, the active sites of which are located on the inner surface of the membrane. In contrast, the catalytic site of the acyl/alkyl‐DHAP reductase, which generates sn‐1‐alkyl‐glycerol‐3‐phosphate, is located on the outer surface. Long‐chain fatty alcohols, which are obligate precursors of ether lipids and wax esters, are biosynthetized by the reduction of the corresponding acyl‐CoAs via the action of an acyl‐CoA reductase. Peroxisome proliferators do not appear to stimulate these enzyme activities specifically. However, we report that feno ‐and ciprofibrate treatments increase six‐fold the palmitoyl‐CoA synthetase mRNA level in the rat liver.

In vivo incorporation of lauric acid into rat adipose tissue triacylglycerols.

An in vivo approach was taken to examine fatty acid esterification in adipose tissue using a coconut oilenriched diet. Rats were fed a diet containing cocounut oil (50% lauric acid) for six weeks. Triacylglycerols from perirenal adipose tissue were fractionated by silver nitrate-thin layer chromatography and, then, preparative gas chromatography. The distribution of 169 triacylglycerol types accounting for 97% of total triacylglycerols was determined. There was evidence for a very high content of mixed triacylglycerols composed of intermediate (12∶0 and 14∶0) and long acyl moieties. No significant differences were observed between the experimental distribution of triacylglycerol types and the random distribution, calculated from the total fatty acid composition. This indicated that most long chain triacylglycerols stored before coconut oil feeding would have been rearranged after the six weeks of coconut oil feeding. The experimental proportion of trilauroylglycerol reached 2%, as expected from its random proportion, and the proportions of dilauroylacylglycerols were slightly higher than the random values. Present results were compared with those previously obtained from triacylglycerols of adipose tissue of rats fed a low-fat standard diet (1,2). From our results and those of other authors, it is suggested that lauric acid is a good substrate forsn-glycero-3-phosphate acyltransferase and diacylglycerol acyltransferase in rat adipose tissue.

Turnover and uptake of double-labelled high-density lipoprotein sphingomyelin in the adult rat

Rat HDL containing [stearic acid-14C, (methyl-3H)choline]sphingomyelin was prepared by incubating labelled sphingomyelin liposomes with serum. HDL was then separated by ultracentrifugation and purified by gel-filtration chromatography. The maximum transfer was reached when 1.5 microliter sphingomyelin was incubated in the presence of 1 ml of serum at 37 degrees C for 1 h. When transfer was limited to a 5-7% increase in HDL mass, no significant change was observed in the HDL electrophoretic pattern, and rats could therefore be injected with this type of HDL under physiological conditions. Plasma radioactivity decay was followed for 24 h, and the recovery of both isotopes in 11 tissues was studied 24 h after the injection. The decay in plasma of both isotopes followed three exponential phases. During the first two phases, both isotopes disappeared with the same velocity (t1/2 = 12.8 and 98-105 min for the first and second phases, respectively). 10 h after injection, 3H had disappeared more slowly than 14C (t1/2 = 862 and 502 min for 3H and 14C, respectively) and 24 h after injection, only 1.5% of 14C and 2.5% of 3H remained in the plasma. This radioactivity was located mainly in HDL (80-85% for 3H and 14C), with a 3H/14C ratio close to that of injected sphingomyelin, and in VLDL, with the same isotopic ratio as that of liver lipids. Some 3H was associated with non-lipoprotein proteins. 17.5% of 3H and 23.4% of 14C were recovered in the liver, 1.6% of each isotope in erythrocytes, and 1.4% of 3H and 0.6% of 14C in kidney. Less than 1% of each isotope was recovered in each of the other tissues. Phosphatidylcholine was the lipid most labelled, and in several tissues sphingomyelin had a 3H/14C ratio close to that of injected sphingomyelin, showing an uptake without prior hydrolysis.

The mouse gene encoding the peroxisomal membrane protein 1‐like protein (PXMP1‐L): cDNA cloning, genomic organization and comparative expression studies1

PXMP1‐L (synonyms: PMP69, P70R) is a peroxisomal protein that belongs to the ABC‐transporter superfamily. Its closest homolog is the peroxisomal membrane protein 1 (PMP70). We have cloned the mouse PXMP1‐L gene. It encodes a 606 amino acid protein. In contrast to the human and the rat, mouse PXMP1‐L is predominantly expressed in the liver. The mouse PXMP1‐L gene consists of 19 exons and spans 21 kb of genomic sequence. No obvious peroxisome proliferator response element has been found in 1.1 kb of the putative promoter region. No coordination of constitutive or fenofibrate‐induced expression of PXMP1‐L with other peroxisomal ABC transporters was observed so that an obligate exclusive heterodimer formation is not likely to occur. The data presented will be particularly useful for the generation of a mouse model defective in PXMP1‐L in order to elucidate the yet unknown function of this protein.

Utilization of High-Density Lipoprotein Sphingomyelin by the Developing and Mature Brain in the Rat

Abstract: Utilization of very long chain saturated fatty acids by brain was studied by injecting 20‐day‐old and adult rats with high‐density lipoprotein containing [stearic or lignoceric acid‐14C, (methyl‐3H)choline]sphingomyelin. Labeling was followed for 24 h. Very small amounts of 14C were recovered in the brain of all rats, and there was no preferential uptake of lignoceric acid. Approximately 20% of the entrapped 14C was located in the form of unchanged sphingomyelin 24 h after injection. This result shows that the rat brain utilizes very little very long chain fatty acids (≥20 C atoms) from high‐density lipoprotein sphingomyelin, even during the myelinating period. The [3H]choline moiety from sphingomyelin was recovered in brain phosphatidylcholine in a higher proportion in comparison with the 14C uptake. The brain 3H increased throughout the studied period in all experiments, but was much higher in the myelinating brain than in the mature brain. From the radioactivity distribution in liver and plasma lipids, it is clear that the choline 3H in the brain originates from either double‐labeled phosphatidylcholine of lipoproteins or tritiated lysophosphatidylcholine bound to albumin, both synthesized by the liver.

Pharmacological Induction of Redundant Genes for a Therapy of X-ALD

X-linked adrenoleukodystrophy (X-ALD) is a recessive neurologic disease with an incidence among males of 1/17 000. Since the identification of the X-ALD gene (ABCD1) ten years ago (Mosser et al 1993), no satisfactory therapy has been available. A close homologue (ABCD2) was then cloned and presented as a putative modifier gene that could account for some of the extreme phenotypic variability of X-ALD (Lombard-Platet et al 1996). The inducibility of Abcd2 by the hypolipidemic drug fenofibrate in the liver of rodents (Albet et al 1997), correlated to a partial normalisation of the biochemical phenotype of X-ALD (Netik et al 1999), opened up the way of a pharmacological therapy of X-ALD. The basis of such a therapy and the results obtained chiefly in rodents will be presented in this chapter.

Copurification of dihydroxyacetone-phosphate acyl-transferase and other peroxisomal proteins from liver of fenofibrate-treated rats.

Dihydroxyacetone-phosphate acyl-transferase (DHAP-AT), a peroxisomal membrane-bound enzyme that catalyzes the first step of ether-glycerolipid synthesis, was purified from liver of rats treated with fenofibrate, a peroxisome proliferator. The protocol first included isolation of peroxisomes, their purification through a discontinuous gradient and solubilization of membranes in CHAPS. DHAP-AT was further purified by four chromatographic steps, namely low-pressure size-exclusion, cation-exchange, hydroxylapatite and chromatofocusing. The chromatofocusing step led to a 4000-fold increase in the specific activity of DHAP-AT with respect to the liver homogenate with a yield of about 0.2%. Trypsin digestion of a 64-kDa protein band upon SDS-PAGE resulted in a peptide sequence unknown in databases. A corresponding degenerated oligonucleotide was used as a probe in Northern blotting, and a transcript of 3.3 kb was detected in some rat tissues. Moreover, the overall procedure allowed co-purification of four major peroxisomal enzymes: urate-oxidase, catalase, multifunctional enzyme and palmitoyl-CoA oxidase, respectively.