Therapy for X-adrenoleukodystrophy: normalization of very long chain fatty acids and inhibition of induction of cytokines by cAMP
1998; Elsevier BV; Volume: 39; Issue: 5 Linguagem: Inglês
10.1016/s0022-2275(20)33878-5
ISSN1539-7262
AutoresKalipada Pahan, Mushfiquddin Khan, Inderjit Singh,
Tópico(s)Adipose Tissue and Metabolism
ResumoX-adrenoleukodystrophy (X-ALD) is an inherited fatty acid metabolic disorder with secondary manifestation of neuroinflammatory disease process. We report that compounds (forskolin, 8-bromo cAMP, and rolipram) that increase cAMP and activate protein kinase A (PKA) were found to stimulate the peroxisomal β-oxidation of lignoceric acid (C24:0) whereas compounds (H-89 and myristoylated PKI) that decrease cAMP and PKA activity inhibited the peroxisomal β-oxidation of lignoceric acid in cultured skin fibroblasts from X-ALD patients. Consistent with the stimulation of β-oxidation of lignoceric acid, activators of PKA normalized the level of very long chain fatty acids (VLCFA) in X-ALD cultured skin fibroblasts. This normalization of VLCFA in X-ALD cells with forskolin, 8-Br cAMP or with rolipram, an inhibitor of cAMP phosphodiesterase, was realized independent of expression of mRNA or protein of the ALD gene, suggesting that cAMP derivatives can correct the metabolic defect in X-ALD fibroblasts without involving the candidate gene for the disease. Because astrocytes and microglia in demyelinating lesions of X-ALD brain express proinflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β), we examined the effect of cAMP derivatives or rolipram on lipopolysaccharide-stimulated rat primary astrocytes and microglia and found that cAMP derivatives and rolipram inhibited the induction of TNF-α and IL-1β in both astrocytes and microglia. The ability of cAMP derivatives and rolipram to block the induction of TNF-α and IL-1β in astrocytes and microglia and to normalize the fatty acid pathogen in skin fibroblasts of x-adrenoleukodystrophy (X-ALD) clearly identify cAMP analogs or rolipram as candidates for potential therapy for X-ALD patients.—Pahan, K., M. Khan, and I. Singh. Therapy for X-adrenoleukodystrophy: normalization of very long chain fatty acids and inhibition of induction of cytokines by cAMP. J. Lipid Res. 1998. 39: 1091–1100. X-adrenoleukodystrophy (X-ALD) is an inherited fatty acid metabolic disorder with secondary manifestation of neuroinflammatory disease process. We report that compounds (forskolin, 8-bromo cAMP, and rolipram) that increase cAMP and activate protein kinase A (PKA) were found to stimulate the peroxisomal β-oxidation of lignoceric acid (C24:0) whereas compounds (H-89 and myristoylated PKI) that decrease cAMP and PKA activity inhibited the peroxisomal β-oxidation of lignoceric acid in cultured skin fibroblasts from X-ALD patients. Consistent with the stimulation of β-oxidation of lignoceric acid, activators of PKA normalized the level of very long chain fatty acids (VLCFA) in X-ALD cultured skin fibroblasts. This normalization of VLCFA in X-ALD cells with forskolin, 8-Br cAMP or with rolipram, an inhibitor of cAMP phosphodiesterase, was realized independent of expression of mRNA or protein of the ALD gene, suggesting that cAMP derivatives can correct the metabolic defect in X-ALD fibroblasts without involving the candidate gene for the disease. Because astrocytes and microglia in demyelinating lesions of X-ALD brain express proinflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β), we examined the effect of cAMP derivatives or rolipram on lipopolysaccharide-stimulated rat primary astrocytes and microglia and found that cAMP derivatives and rolipram inhibited the induction of TNF-α and IL-1β in both astrocytes and microglia. The ability of cAMP derivatives and rolipram to block the induction of TNF-α and IL-1β in astrocytes and microglia and to normalize the fatty acid pathogen in skin fibroblasts of x-adrenoleukodystrophy (X-ALD) clearly identify cAMP analogs or rolipram as candidates for potential therapy for X-ALD patients.—Pahan, K., M. Khan, and I. Singh. Therapy for X-adrenoleukodystrophy: normalization of very long chain fatty acids and inhibition of induction of cytokines by cAMP. J. Lipid Res. 1998. 39: 1091–1100. X-linked adrenoleukodystrophy (X-ALD), an inherited peroxisomal disorder, is characterized by progressive demyelination and adrenal insufficiency (1Singh I. Biochemistry of peroxisomes in health and disease.Mol. Cell. Biochem. 1997; 167: 1-29Google Scholar, 2Moser H.W. Moser A.E. Singh I. O'Neill B.P. Adrenoleukodystrophy: survey of 303 cases, biochemistry, diagnosis, and therapy.Ann. Neurol. 1984; 16: 628-641Google Scholar). It is the most common peroxisomal disorder affecting between 1/15,000 to 1/20,000 boys and manifests with different degrees of neurological disability. The onset of childhood X-ALD, the major form of X-ALD, is between the ages of 4 to 8 and then death occurs within the next 2 to 3 years. As yet no proven therapy improves or changes the course of the disease process in X-ALD patients. All forms of X-ALD accumulate pathognomonic amounts of saturated very long chain fatty acids (VLCFA). In fact, levels of VLCFA have been used as a tool for both prenatal and post-natal diagnosis (1Singh I. Biochemistry of peroxisomes in health and disease.Mol. Cell. Biochem. 1997; 167: 1-29Google Scholar, 2Moser H.W. Moser A.E. Singh I. O'Neill B.P. Adrenoleukodystrophy: survey of 303 cases, biochemistry, diagnosis, and therapy.Ann. Neurol. 1984; 16: 628-641Google Scholar). A number of laboratories, including ours, have previously demonstrated that VLCFA are mainly and preferentially β-oxidized in peroxisomes and that VLCFA in X-ALD accumulate because of a defect in their oxidation in peroxisomes (3Singh I. Moser A.E. Goldfischer S. Moser H.W. Lignoceric acid is oxidized in peroxisome: implication for the Zellweger cerebro-hepato-renal syndrome and adrenoleukodystrophy.Proc. Natl. Acad. Sci. USA. 1984; 81: 4203-4207Google Scholar, 4Hashmi M. Stanley W. Singh I. Lignoceroyl-CoASH ligase: enzyme defect in fatty acid β-oxidation system in X-linked childhood adrenoleukodystrophy.FEBS Lett. 1986; 196: 247-250Google Scholar, 5Lageweg W. Sykes J.E.C. Lopes-Cardozo M. Wanders R.J.A. Oxidation of very long chain fatty acids in rat brain: cerotic acid is β-oxidized exclusively in rat brain peroxisomes.Biochim. Biophys. Acta. 1991; 1085: 381-384Google Scholar). Studies with total cellular homogenates and subsequent studies with purified subcellular organelles from cultured skin fibroblasts of X-ALD and control directly demonstrated the deficiency in VLC fatty acyl-CoA ligase in peroxisomes (6Lazo O. Contreras M. Hashmi M. Stanley W. Irazu C. Singh I. Peroxisomal lignoceroyl-CoA ligase deficiency in childhood adrenoleukodystrophy and adrenomyeloneuropathy.Proc. Natl. Acad. Sci. USA. 1988; 85: 7647-7651Google Scholar, 7Lazo O. Contreras M. Bhusan A. Stanley W. Singh I. Adrenoleukodystrophy: impaired oxidation of fatty acids due to peroxisomal lignoceroyl-CoA ligase deficiency.Arch. Biochem. Biophys. 1989; 270: 722-728Google Scholar). While these metabolic studies indicated lignoceroyl-CoA ligase gene as an X-ALD gene, positional cloning studies led to the identification of a gene that codes for a protein (ALDP), an 84 kDa protein that migrates as a 75 kDa protein in SDS-PAGE, with significant homology with the ATP-binding cassette of the super family of transporters (8Mosser J. Douar A.M. Sarde C.O. Kioschis P. Feil R. Moser H. Poustka A.M. Mandel J.L. Aubourg P. Putative X-linked adrenoleukodystrophy gene shares unexpected homology with ABC transporters.Nature. 1993; 361: 726-730Google Scholar). Studies from our laboratory (9Contreras M. Mosser J. Mandel J.L. Aubourg P. Singh I. The protein coded by the X-adrenoleukodystrophy gene is a peroxisomal integral membrane protein.FEBS Lett. 1994; 344: 211-215Google Scholar) and those of others (10Watkins P.A. Gould S.J. Smith M.A. Braiterman L.T. Wei H.M. Kok F. Moser A.B. Moser H.W. Smith K.D. Altered expression of ALDP in X-linked adrenoleukodystrophy.Am. J. Hum. Genet. 1995; 57: 292-301Google Scholar) demonstrated that ALDP is a peroxisomal membrane protein component and that the ATP-binding domain of ALDP, approximately 43 kDa, is oriented toward the cytoplasmic surface of the peroxisomal membrane (11Contreras M. Sengupta T.K. Sheikh F. Aubourg P. Singh I. Topology of ATP-binding domain of adrenoleukodystrophy gene product in peroxisomes.Arch. Biochem. Biophys. 1996; 334: 369-379Google Scholar). The normalization of fatty acids in X-ALD cells after transfection of the X-ALD gene (12Cartier N. Lopez J. Moullier P. Rocchiccioli F. Rolland M.O. Jorge P. Mosser J. Mandel J.L. Bougneres P.F. Danos O. Aubourg P. Retroviral-mediated gene transfer corrects very-long-chain fatty acid metabolism in adrenoleukodystrophy fibroblasts.Proc. Natl. Acad. Sci. USA. 1995; 92: 1674-1678Google Scholar) supports a role for ALDP in fatty acid metabolism, however, the precise function of ALDP in the metabolism of VLCFA is not known at the present time. Mutations (missense or nonsense) or gene deletions have been detected in 80% of the X-ALD patients and these mutations were distributed over the whole protein-coding region, except exon 10, and nearly each patient has a different mutation (13Ligtenberg M. Kemp S. Sarde C.O. van Geel B.M. Kleijer W.J. Barth P.G. Mandel J.L. van Oost B.A. Bolhuis P.A. Spectrum of mutations in the gene encoding the adrenoleukodystrophy protein.Am. J. Hum. Genet. 1995; 56: 44-50Google Scholar, 14Krasemann E.W. Meier V. Korenke G.C. Hunneman D.H. Hanefeld F. Identification of mutations in the ALD-gene of 20 families with adrenoleukodystrophy/adrenomyeloneuropathy.Hum. Genet. 1996; 97: 194-197Google Scholar, 15Moser H.W. Clinical and therapeutic aspects of adrenoleukodystrophy and adrenomyeloneuropathy.J. Neuropathol. Exp. Neurol. 1995; 54: 740-744Google Scholar). Moreover, no relationship could be established between the genotype and the severity of the disease as the same mutation is known to give different types of phenotype (1Singh I. Biochemistry of peroxisomes in health and disease.Mol. Cell. Biochem. 1997; 167: 1-29Google Scholar, 15Moser H.W. Clinical and therapeutic aspects of adrenoleukodystrophy and adrenomyeloneuropathy.J. Neuropathol. Exp. Neurol. 1995; 54: 740-744Google Scholar). Similar to other genetic diseases affecting the central nervous system, the gene therapy in X-ALD does not seem to be a real option in the near future and in the absence of such a treatment a number of therapeutic applications have been investigated (1Singh I. Biochemistry of peroxisomes in health and disease.Mol. Cell. Biochem. 1997; 167: 1-29Google Scholar, 15Moser H.W. Clinical and therapeutic aspects of adrenoleukodystrophy and adrenomyeloneuropathy.J. Neuropathol. Exp. Neurol. 1995; 54: 740-744Google Scholar). Adrenal insufficiency associated with X-ALD responds readily to steroid replacement therapy, however, there is as yet no proven therapy for neurological disability (15Moser H.W. Clinical and therapeutic aspects of adrenoleukodystrophy and adrenomyeloneuropathy.J. Neuropathol. Exp. Neurol. 1995; 54: 740-744Google Scholar). Two forms of therapies are presently under current investigation. Dietary therapy with "Lorenzo's oil" does normalize the plasma levels of VLCFA, however, it does not seem to improve the clinical status of the X-ALD patients (15Moser H.W. Clinical and therapeutic aspects of adrenoleukodystrophy and adrenomyeloneuropathy.J. Neuropathol. Exp. Neurol. 1995; 54: 740-744Google Scholar, 16Rizzo W.B. Watkins P.A. Philips M.W. Cranin D. Campbell B. Avigan J. Adrenoleukodystrophy: oleic acid lowers fibroblasts saturated C22–26 fatty acids.Neurology. 1986; 36: 357-361Google Scholar, 17Rizzo W.B. Leshne R.T. Odone A. Dammann A.L. Craft D.A. Jensen M.E. Jennings S.S. Davis S. Jaitly R. Sgro J.A. Dietery erucic acid therapy for X-linked adrenoleukodystrophy.Neurology. 1989; 39: 1415-1422Google Scholar). These results, in part, may be due to the fact that the fatty acid composition of the brain is not normalized because of a failure of erucic acid to enter the brain in significant quantity (15Moser H.W. Clinical and therapeutic aspects of adrenoleukodystrophy and adrenomyeloneuropathy.J. Neuropathol. Exp. Neurol. 1995; 54: 740-744Google Scholar). Bone marrow therapy also appears to be of limited value because of the complexity of the protocol and of insignificant efficacy in improving the clinical status of the patient (15Moser H.W. Clinical and therapeutic aspects of adrenoleukodystrophy and adrenomyeloneuropathy.J. Neuropathol. Exp. Neurol. 1995; 54: 740-744Google Scholar). Because X-ALD is a metabolic disorder of VLCFA that eventually leads to an inflammatory bilateral demyelination with marked activation of microglia and astrocytes and accumulation of proinflammatory cytokines (TNF-α and IL-1β) and extracellular matrix proteins (18Powers J.M. Liu Y. Moser A.B. Moser H.W. The inflammatory myelinopathy of adrenoleukodystrophy: cells, effector molecules, and pathogenetic implications.J. Neuropathol. Exp. Neurol. 1992; 51: 630-643Google Scholar, 19McGuinness M.C. Griffin D.E. Raymond G.V. Washington C.A. Moser H.W. Smith K.D. Tumor necrosis factor-α and X-linked adrenoleukodystrophy.J. Neuroimmunol. 1995; 61: 161-169Google Scholar), we searched for a therapy that would normalize the VLCFA and inhibit the induction of proinflammatory cytokines by astrocytes and microglia. The studies described in this paper demonstrate that the compounds that increase the intracellular levels of cAMP and the activity of protein kinase A (PKA) normalize the levels of VLCFA, possibly by increasing the peroxisomal activity for β-oxidation of VLCFA. Moreover, the same compounds also inhibit the induction of TNF-α and IL-1β in lipopolysaccharide (LPS)-stimulated astrocytes and microglia. These observations demonstrate the therapeutic potential of compounds that increase the activity of PKA in correction of the metabolic defect and inhibition of the neuroinflammatory disease process in X-ALD. DMEM and bovine calf serum were from GIBCO. Forskolin, 1,9-dideoxyforskolin, 8-Br cAMP, S(p)-cAMP, H-89, rp-cAMP and rolipram were obtained from Biomol, Plymouth Meeting, PA. C18:0-CoA, NADPH and N-ethylmaleimide were from Sigma (St. Louis, MO). [2-14C]malonyl-CoA and K14CN (52 mCi/mmol) were purchased from DuPont-New England Nuclear. [1-14C]lignoceric acid was synthesized by treatment of n-tricosanoyl bromide with K14CN as described previously (20Hoshi M. Kishimoto Y. Synthesis of cerebronic acid from lignoceric acid by rat brain preparation. Some properties and distribution of the hydroxylation system.J. Biol. Chem. 1973; 248: 4123-4130Google Scholar). The enzyme activity of [1-14C]lignoceric acid β-oxidation to acetate was measured in intact cells suspended in Hank's buffered salt solution (HBSS). Briefly, the reaction mixture in 0.25 ml of HBSS contained 50–60 μg of protein and 6 μm [1-14C]lignoceric acid. Fatty acids were solubilized with α-cyclodextrin, and β-oxidation of [1-14C]lignoceric acid was carried out as described previously (3Singh I. Moser A.E. Goldfischer S. Moser H.W. Lignoceric acid is oxidized in peroxisome: implication for the Zellweger cerebro-hepato-renal syndrome and adrenoleukodystrophy.Proc. Natl. Acad. Sci. USA. 1984; 81: 4203-4207Google Scholar, 6Lazo O. Contreras M. Hashmi M. Stanley W. Irazu C. Singh I. Peroxisomal lignoceroyl-CoA ligase deficiency in childhood adrenoleukodystrophy and adrenomyeloneuropathy.Proc. Natl. Acad. Sci. USA. 1988; 85: 7647-7651Google Scholar). The reaction was stopped after 1 h with 0.625 ml of 1 m KOH in methanol, and the denatured protein was removed by centrifugation. The supernatant was incubated at 60°C for 1 h, neutralized with 0.125 ml of 6 N HCl, and partitioned with chloroform and methanol. Radioactivity in the upper phase is an index of [1-14C]lignoceric acid oxidized to acetate. Cultured skin fibroblasts from patients with X-adrenoleukodystrophy were obtained from NIGMS Human Genetic Mutant Cell Repository, USA. These studies were approved by institutional approval (AR# 1128). Cells were incubated for 15 min at 37°C under isotonic conditions in HBSS with [1-14C]lignoceric acid (6 μm) solubilized with α-cyclodextrin as described earlier (3Singh I. Moser A.E. Goldfischer S. Moser H.W. Lignoceric acid is oxidized in peroxisome: implication for the Zellweger cerebro-hepato-renal syndrome and adrenoleukodystrophy.Proc. Natl. Acad. Sci. USA. 1984; 81: 4203-4207Google Scholar, 5Lageweg W. Sykes J.E.C. Lopes-Cardozo M. Wanders R.J.A. Oxidation of very long chain fatty acids in rat brain: cerotic acid is β-oxidized exclusively in rat brain peroxisomes.Biochim. Biophys. Acta. 1991; 1085: 381-384Google Scholar). Then cells were separated from the incubation medium by centrifugation through an organic layer of brominated hydrocarbons (21Singh I. Lazo O. Dhaunsi G.S. Contreras M. Transport of fatty acids into human and rat peroxisomes: differential transport of palmitic and lignoceric acids and its implication to X-adrenoleukodystrophy.J. Biol Chem. 1992; 267: 13306-13313Google Scholar). This was performed in micro tubes (1.5 ml) containing 50 μl of 0.25 m sucrose in HBSS (as cushion), an organic layer (400 μl) consisting of a mixture of bromododecane and bromodecane (7:4, v/v), and an upper layer (500 μl) of cells in HBSS. Cell extracts were assayed for PKA activity as described (22Graves L.M. Bornfeildt K.E. Raines E.W. Potts B.C. Macdonald S.G. Ross R. Krebs E.G. Protein kinase A antagonizes platelet-derived growth factor-induced signaling by mitogen-activated protein kinase in human arterial smooth muscle cells.Proc. Natl. Acad. Sci. USA. 1993; 90: 10300-10304Google Scholar, 23Pahan K. Namboodiri A.M.S. Sheikh F.G. Smith B.T. Singh I. Increasing cAMP attenuates induction of inducible nitric oxide synthase in rat primary astrocytes.J. Biol. Chem. 1997; 272: 7786-7791Google Scholar) by measuring the phosphorylation of kemptide (0.17 mm) in the presence or absence of PKI peptide (15 μm). PKA activity was calculated as the amount of kemptide phosphorylated in the absence of PKI peptide minus that phosphorylated in the presence of PKI peptide. The fatty acid elongation activity was assayed by the method of Tsuji et al. (24Tsuji S. Ohno T. Miyatake T. Suzuki A. Yamakawa T. Fatty acid elongation activity in fibroblasts from patients with adrenoleukodystrophy (ALD).J. Biochem. 1984; 96: 1241-1247Google Scholar). Briefly, the assay mixture contained 100 mm potassium phosphate (pH 7.2), 0.5 mm NADPH, 0.05 mm [2-14C]malonyl-CoA, 1 mm N-ethyl maleimide, and 50–60 μg of protein in a total volume of 0.25 ml. The concentration of C18:0-CoA was 1 μm. The reaction was started at 37°C by the addition of total homogenate and stopped by the addition of 1.25 ml of 10% (w/v) KOH after 30 min incubation. After saponification at 100°C for 30 min, the solutions were acidified with 1 ml of 4 N HCl and fatty acids were extracted with 2.5 ml of n-pentane three times. The radioactivities incorporated into fatty acids were measured with a liquid scintillation counter. Fatty acid methyl ester (FAME) was prepared as described previously by Lepage and Roy (25Lepage G. Roy C.C. Direct transesterification of all classes of lipids in one-step reaction.J. Lipid Res. 1986; 27: 114-120Google Scholar) with modifications. Fibroblast cells, suspended in HBSS, were disrupted by sonication to form a homogeneous solution. An aliquot (200 μl) of this solution was transferred to a glass tube and 5 μg heptacosanoic (27:0) acid was added as internal standard and lipids were extracted by Folch partition. Fatty acids were transesterified with acetyl chloride (200 μl) in the presence of methanol and benzene (4:1) for 2 h at 100°C. The solution was cooled down to room temperature followed by addition of 5 ml 6% potassium carbonate solution at ice-cooled temperature. Isolation and purification of FAME were carried out as detailed by Dacremont, Cocquyt, and Vincent (26Dacremont G. Cocquyt G. Vincent G. Measurement of very long chain fatty acids, phytanic acid and pristanic acid in plasma and cultured fibroblasts by gas chromatography.J. Inher. Metab. Dis. 1995; 18: 76-83Google Scholar). Purified FAME, suspended in chloroform, were analyzed by a gas chromatograph GC-15A attached with a Chromatopac C-R3A integrator from Shimadzu Corporation. The membranes were prepared as described previously (11Contreras M. Sengupta T.K. Sheikh F. Aubourg P. Singh I. Topology of ATP-binding domain of adrenoleukodystrophy gene product in peroxisomes.Arch. Biochem. Biophys. 1996; 334: 369-379Google Scholar). Briefly, the post-nuclear fraction was diluted with an ice-cold solution of 0.1 m sodium carbonate, 30 mm iodoacetamide, pH 11.5. After 30 min incubation at 4°C, the membranes were sedimented by ultracentrifugation. The sedimented membranes were electrophoresed in 7.5% sodium dodecylsulfate-polyacrylamide gel, transferred to PVDF membranes and immunoblotted with antibodies against ALDP as described (11Contreras M. Sengupta T.K. Sheikh F. Aubourg P. Singh I. Topology of ATP-binding domain of adrenoleukodystrophy gene product in peroxisomes.Arch. Biochem. Biophys. 1996; 334: 369-379Google Scholar). Cultured skin fibroblasts were taken from culture flasks directly by adding Ultraspec-II RNA reagent (Biotecx Laboratories Inc.) and total RNA was isolated according to the manufacturer's protocol. Twenty micrograms of RNA from each sample was electrophoretically resolved on 1.2% denaturing formaldehyde-agarose gel, transferred to nylon membrane, and cross-linked using UV Stratalinker (Stratagene, San Diego, CA). Full-length ALDP cDNA was kindly provided by Dr. Patrick Aubourg, INSERM, Hospital Saint-Vincent-de-Paul, Paris, France. 32P-labeled cDNA probes were prepared according to the instructions provided with Ready-To-Go DNA labeling kit (Pharmacia Biotech). Northern blot analysis was performed essentially as described for Express Hyb Hybridization solution (Clontech) at 68°C. Actin cDNA probe was used as standard for comparing hybridization signals. Astrocytes were prepared from rat cerebral tissue as described earlier (23Pahan K. Namboodiri A.M.S. Sheikh F.G. Smith B.T. Singh I. Increasing cAMP attenuates induction of inducible nitric oxide synthase in rat primary astrocytes.J. Biol. Chem. 1997; 272: 7786-7791Google Scholar, 27McCarthy K. DeVellis J. Preparation of separate astroglial and oligodendroglial cultures from rat cerebral tissue.J. Cell Biol. 1980; 85: 890-902Google Scholar). Microglial cells were isolated from mixed glial cultures according to the procedure of Giulian and Baker (28Giulian D. Baker T.J. Characterization of amoebid microglia isolated from developing mammalian brain.J. Neurosci. 1986; 6: 2163-2178Google Scholar). For the induction of cytokine production, cells were stimulated with LPS in serum-free condition. Cells were stimulated with LPS in serum-free media for 24 h in the presence or absence of forskolin or rolipram, and concentrations of TNF-α and IL-1β were measured in culture supernatants by using high-sensitivity enzyme-linked immunosorbent assay (R&D Systems) according to the manufacturer's instructions. First, we studied the effect of cAMP derivatives on lignoceric acid β-oxidation in control human fibroblasts. Cultured skin fibroblasts were treated with different activators and inhibitors of protein kinase A (PKA) and tested for β-oxidation of lignoceric acid. It is apparent from Table 1 that compounds known to increase cAMP (forskolin and 8-Br-cAMP) stimulated lignoceric acid β-oxidation whereas compounds known to decrease cAMP (H-89 and myristoylated PKI) inhibited lignoceric acid β-oxidation in control skin fibroblasts. The inactive analogue of forskolin, 1,9-dideoxyforskolin, was ineffective in stimulating β-oxidation (Table 1). These results suggest that PKA has a positive modulatory role on lignoceric acid β-oxidation. As the β-oxidation of lignoceric acid is impaired in X-ALD patients, we studied the effect of different activators and inhibitors of PKA on lignoceric acid β-oxidation in cultured skin fibroblasts of X-ALD. Figure 1 shows that the compounds (forskolin, 8-bromo cAMP and rolipram) known to increase intracellular cAMP stimulated lignoceric acid β-oxidation (Fig. 1A) and activated the PKA activity (Fig. 1C). On the other hand, β-oxidation of lignoceric acid was inhibited by PKA inhibitors (H-89 and myristoylated PKI). A combination of forskolin (activator of PKA) and H-89 or myristoylated PKI (inhibitors of PKA) had relatively little effect on the activation of PKA as well as on the β-oxidation of lignoceric acid. These observations indicate that β-oxidation of lignoceric acid is modulated by cAMP and PKA. However, in contrast to the effects on β-oxidation of lignoceric acid, activators of PKA inhibited the fatty acid chain elongation and inhibitors of PKA stimulated this activity in X-ALD fibroblasts (Fig. 1B). The increase in β-oxidation of lignoceric acid by forskolin (Fig. 2A) and its inhibition by H-89 (Fig. 2B) were dose-dependent. To understand the mechanism of cAMP-mediated stimulation of lignoceric acid β-oxidation, fibroblasts of X-ALD were treated with cAMP analogs, and the transport of lignoceric acid into intact cells and β-oxidation of lignoceric acid in cell homogenates of X-ALD were measured. Similar to the modulation of lignoceric acid β-oxidation, activators of PKA also stimulated the transport of lignoceric acid into ALD cells by more than 2-fold whereas inhibitors of PKA inhibited the transport of lignoceric acid by 40–50% (data not shown). Stimulation of lignoceric acid β-oxidation in cell homogenates of ALD fibroblasts as well as in cell suspension (Fig. 1A) suggests that increase in β-oxidation may not be due to an intracellular increase of substrate concentration but possibly to stimulation of the enzyme system for oxidation of lignoceric acid. In the cell, fatty acids are oxidized by mitochondrial and peroxisomal β-oxidation enzyme. Etomoxir, an inhibitor of mitochondrial β-oxidation of fatty acids (29Mannaerts G.P. Debeer L.J. Thomas J. De Schepper P.J. Mitochondrial and peroxisomal fatty acid oxidation in liver homogenates and isolated hepatocytes from control and clofibrate-treated rats.J. Biol. Chem. 1979; 254: 4585-4595Google Scholar), had no effect on cAMP-mediated stimulation of lignoceric acid β-oxidation (data not shown) suggesting that the observed stimulation of lignoceric acid β-oxidation was a peroxisomal function. The increase in β-oxidation and transport of lignoceric acid but the decrease in fatty acid chain elongation with the increase in cAMP level and PKA activity and the decrease in β-oxidation and transport of lignoceric acid but the increase in fatty acid chain elongation with the decrease in cAMP level and PKA activity clearly delineate cAMP and cAMP-dependent protein kinase A as important regulators of the metabolism of VLCFA.TABLE 1.Effects of different agonists and antagonists of PKA on β-oxidation of lignoceric acid in control human fibroblastsTreatmentLignoceric acid β-oxidation(pmol/h/mg protein)Control565.2 ± 48.3Forskolin885.3 ± 62.11,9 Dideoxy forskolin571.4 ± 39.68-Br-cAMP872.0 ± 53.7H-89405.6 ± 44.1Myristoylated PKI432.3 ± 46.5Cells were treated for 72 h in serum-containing DMEM with the listed reagents. β-oxidation of lignoceric acid was measured as described in Material and Methods. Media were replaced every 24 h with the addition of fresh reagents. Concentrations of reagents were: forskolin, 4 μm; 1,9 dideoxy forskolin; 4 μm 8-Br-cAMP, 50 μm; H-89, 1 μm; myristoylated PKI, 0.2 μm. Data are means ± SD of three different experiments. Open table in a new tab Fig. 2.Forskolin stimulates whereas H-89 inhibits the β-oxidation of lignoceric acid in cultured skin fibroblasts of X-ALD in a dose-dependent manner. Cells were incubated in serum-containing DMEM with different concentrations of forskolin (A) or H-89 (B) for 72 h. Every 24 h, media were replaced with the addition of fresh reagents. β-Oxidation of lignoceric acid was measured in cell suspension as mentioned in Methods (•, experiment 1; ○, experiment 2).View Large Image Figure ViewerDownload (PPT) Cells were treated for 72 h in serum-containing DMEM with the listed reagents. β-oxidation of lignoceric acid was measured as described in Material and Methods. Media were replaced every 24 h with the addition of fresh reagents. Concentrations of reagents were: forskolin, 4 μm; 1,9 dideoxy forskolin; 4 μm 8-Br-cAMP, 50 μm; H-89, 1 μm; myristoylated PKI, 0.2 μm. Data are means ± SD of three different experiments. Because cAMP derivatives increase β-oxidation of lignoceric acid and decrease fatty acid chain elongation, we examined the effect of cAMP derivatives on the level of VLCFA in X-ALD fibroblasts. Treatment of X-ALD fibroblasts with 4 μm forskolin for different time periods (days) resulted in a time-dependent increase in oxidation of lignoceric acid and a time-dependent decrease in the ratios of C26:0/C22:0 and C24:0/C22:0 as shown in Fig. 3. Within 12 to 15 days of treatment, the ratios of C26:0/C22:0 and C24:0/C22:0 in X-ALD fibroblasts decreased to the normal level. The C26:0/C22:0 and C24:0/C22:0 values in control fibroblasts in these culture conditions were 0.04 ± 0.01 (n=8) and 1.32 ± 0.20 (n=8), respectively. This decrease in the ratios of C26:0/C22:0 and C24:0/C22:0 was also associated with the decrease in the absolute amounts of C24:0 and C26:0 whereas no significant change was observed in the levels of C22:0 (behenoic acid) (data not shown). To decipher the possible mechanism of this dramatic decrease of VLCFA, we treated X-ALD fibroblasts with different activators of PKA (forskolin, 8-Br-cAMP, and rolipram) for 15 days and analyzed the level of VLCFA. As shown in Fig. 4, the treatment of X-ALD fibroblasts with compounds known to increase intracellular cAMP lowered the ratios of C26:0/C22:0 and C24:0/C22:0 to the normal lev
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