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Decreasing the expression of very long-chain acyl-CoA synthetase 3 (ACSVL3) in U87MG glioblastoma cells by either RNA interference or genomic knockout (KO) significantly decreased their growth rate in culture, as well as their ability to form rapidly growing tumors in mice. U87-KO cells grew at a 9-fold slower rate than U87MG cells. When injected subcutaneously in nude mice, the tumor initiation frequency of U87-KO cells was 70% of that of U87MG cells, and the average growth rate of tumors that did form was decreased by 9-fold. Two hypotheses to explain the decreased growth rate of KO cells were investigated. Lack of ACSVL3 could reduce cell growth either by increasing apoptosis, or via effects on the cell cycle. We examined intrinsic, extrinsic, and caspase-independent apoptosis pathways; none were affected by lack of ACSVL3. However, significant differences in the cell cycle were seen in KO cells, suggesting arrest in S-phase. Levels of cyclin-dependent kinases 1, 2, and 4 were elevated in U87-KO cells, as were regulatory proteins p21 and p53 that promote cell cycle arrest. In contrast, lack of ACSVL3 reduced the level of the inhibitory regulatory protein p27. γ-H2AX, a marker of DNA double strand breaks, was elevated in U87-KO cells, while pH3, a mitotic index marker, was reduced. Previously reported alterations in sphingolipid metabolism in ACSVL3-depleted U87 cells may explain the effect of KO on cell cycle. These studies reinforce the notion that ACSVL3 is a promising therapeutic target in glioblastoma.

Supplementary Figure 2 from Acyl-CoA Synthetase VL3 Knockdown Inhibits Human Glioma Cell Proliferation and Tumorigenicity

Supplementary Figure 1 from Acyl-CoA Synthetase VL3 Knockdown Inhibits Human Glioma Cell Proliferation and Tumorigenicity

The contribution of lipid metabolic pathways to malignancy is poorly understood. Expression of the fatty acyl-CoA synthetase ACSVL3 was found to be markedly elevated in clinical malignant glioma specimens but nearly undetectable in normal glia. ACSVL3 levels correlated with the malignant behavior of human glioma cell lines and glioma cells propagated as xenografts. ACSVL3 expression was induced by the activation of oncogenic receptor tyrosine kinases (RTK) c-Met and epidermal growth factor receptor. Inhibiting c-Met activation with neutralizing anti–hepatocyte growth factor monoclonal antibodies reduced ACSVL3 expression concurrent with tumor growth inhibition in vivo. ACSVL3 expression knockdown using RNA interference, which decreased long-chain fatty acid activation, inhibited anchorage-dependent and anchorage-independent glioma cell growth by ∼70% and ∼90%, respectively. ACSVL3-depleted cells were less tumorigenic than control cells, and subcutaneous xenografts grew ∼60% slower than control tumors. Orthotopic xenografts produced by ACSVL3-depleted cells were 82% to 86% smaller than control xenografts. ACSVL3 knockdown disrupted Akt function as evidenced by RTK-induced transient decreases in total and phosphorylated Akt, as well as glycogen synthase kinase 3β, via a caspase-dependent mechanism. Expressing constitutively active myr-Akt rescued cells from the anchorage-dependent and anchorage-independent growth inhibitory effects of ACSVL3 depletion. These studies show that ACSVL3 maintains oncogenic properties of malignant glioma cells via a mechanism that involves, in part, the regulation of Akt function. [Cancer Res 2009;69(24):9175–82]

Supplementary Figure Legends 1-6 from Acyl-CoA Synthetase VL3 Knockdown Inhibits Human Glioma Cell Proliferation and Tumorigenicity

Supplementary Figure 4 from Acyl-CoA Synthetase VL3 Knockdown Inhibits Human Glioma Cell Proliferation and Tumorigenicity

Supplementary Figure 6 from Acyl-CoA Synthetase VL3 Knockdown Inhibits Human Glioma Cell Proliferation and Tumorigenicity

Supplementary Methods from Acyl-CoA Synthetase VL3 Knockdown Inhibits Human Glioma Cell Proliferation and Tumorigenicity

Supplementary Figure 3 from Acyl-CoA Synthetase VL3 Knockdown Inhibits Human Glioma Cell Proliferation and Tumorigenicity

By activating fatty acids to their CoA derivatives, acyl-CoA synthetases (ACS) play an essential role in fatty acid metabolism. We previously identified ACSF2 as an ACS that was phylogenetically distinct from known families of short-chain, medium-chain, long-chain, very long-chain, and bubblegum ACSs. Functionally, ACSF2 preferentially activated medium-chain fatty acids. In this work, we provide further characterization of this unique ACS. ACSF2 mRNA expression was found in most tissues, although immunohistochemical analysis revealed differences in protein expression between various cell types in each tissue. Endogenous ACSF2 was found in the Golgi region in Neuro2a and P19 cells, and disruption of the Golgi in Neuro2a cells with nocodazole disrupted ACSF2 localization. In contrast, MA-10, HepG2, and skin fibroblasts had a mitochondrial ACSF2 localization. ACSF2 activated saturated fatty acids containing 6 to 10 carbons when overexpressed in COS-1 cells. The Kmapp for C10:0 was 24.4 μM and Vmaxapp was 385 nmol/20min/mg COS cell protein. Knockdown by RNA interference revealed that ACSF2 was responsible for most of the medium-chain ACS activity in Neuro2a cells. A lysine residue critical for activity in bacterial short-chain ACSs was found in ACSF2, and mutation of this residue to alanine abolished enzyme activity. Neurite outgrowth results when Neuro2a cells are induced to differentiate with retinoic acid, and ACSF2 migrated to nodes and points of neurite-neurite contact along with the presynaptic marker, synaptophysin. ACSF2-deficient Neuro2a cells showed significantly blunted neurite outgrowth in response to retinoic acid. These results suggest that this medium-chain ACS may play an important role in neuronal development.

Long-chain fatty acids are an important source of energy in muscle and heart where the acyl-CoA dehydrogenases (ACADs) participate in consecutive cycles of β-oxidation to generate acetyl-CoA and reducing equivalents for generating energy. However, the role of long-chain fatty acid oxidation in the brain and other tissues that do not rely on fat for energy is poorly understood. Here we characterize two new ACADs, ACAD10 and ACAD11, both with significant expression in human brain. ACAD11 utilizes substrates with primary carbon chain lengths between 20 and 26, with optimal activity towards C22CoA. The combination of ACAD11 with the newly characterized ACAD9 accommodates the full spectrum of long chain fatty acid substrates presented to mitochondrial β-oxidation in human cerebellum. ACAD10 has significant activity towards the branched-chain substrates R and S, 2 methyl-C15-CoA and is highly expressed in fetal but not adult brain. This pattern of expression is similar to that of LCAD, another ACAD previously shown to be involved in long branched chain fatty acid metabolism. Interestingly, the ACADs in human cerebellum were found to have restricted cellular distribution. ACAD9 was most highly expressed in the granular layer, ACAD11 in the white matter, and MCAD in the molecular layer and axons of specific neurons. This compartmentalization of ACADs in the human central nerve system, suggests that β-oxidation in cerebellum participates in different functions other than generating energy, for example, the synthesis and/or degradation of unique cellular lipids and catabolism of aromatic amino acids, compounds that are vital to neuronal function.

X-linked adreno-leukodystrophy is a progressive, systemic peroxisomal disorder that affects primarily nervous system myelin and axons as well as the adrenal cortex. Several divergent clinical phenotypes can occur in the same family; thus, there is no correlation between the clinical phenotype and the mutation in the ABCD1 gene in this disease. The most urgent and unresolved clinical issue is the fulminant inflammatory (immune) demyelination of the central nervous system in which a variety of cellular participants, cytokines, and chemokines are noted. A knockout mouse model exhibits mitochondrial deficits and axonal degeneration, but not inflammatory demyelination. To determine whether oxidative stress and damage might play a pathogenic role, we assessed standard biochemical and immunohistochemical markers of such activity both in our knockout mouse model and patients. We find that oxidative stress, as judged by increased immunoreactivity for the mitochondrial manganese-superoxide dismutase, is present in the knockout mouse liver, adrenal cortex, and renal cortex, tissues that normally express high levels of ABCD1 but no evidence of oxidative damage. The brain does not exhibit either oxidative stress or damage. On the other hand, both the human adrenal cortex and brain show evidence of oxidative stress (e.g. hemoxygenase-1 and manganese-superoxide dismutase) and oxidative damage, particularly from lipid peroxidation (4-hydroxynonenal and malondialdehyde). The presence of nitrotyrosylated proteins is strong circumstantial evidence for the participation of the highly toxic peroxynitrite molecule, whereas the demonstration of interferon gamma and interleukin-12 is indicative of a TH1 response in the inflammatory demyelinative lesions of the cerebral phenotype. These differences between the adreno-leukodystrophy mouse and human patients are intriguing and may provide a clue to the phenotypic divergence in this disease.

The family of proteins that includes very long-chain acyl-CoA synthetases (ACSVL) consists of six members. These enzymes have also been designated fatty acid transport proteins. We cloned full-length mouse Acsvl3 cDNA and characterized its protein product ACSVL3/fatty acid transport protein 3. The predicted amino acid sequence contains two highly conserved motifs characteristic of acyl-CoA synthetases. Northern blot analysis revealed that the mouse Acsvl3 mRNA is highly expressed in adrenal gland, testis, and ovary, with lower expression in the brain of adult mice. A developmental Northern blot revealed that Acsvl3 mRNA levels were significantly higher in embryonic mouse brain (embryonic days 12-14) than in newborn or adult mice, suggesting a possible role in nervous system development. Immunohistochemistry revealed high ACSVL3 expression in adrenal cortical cells, spermatocytes and interstitial cells of the testis, theca cells of the ovary, cerebral cortical neurons, and cerebellar Purkinje cells. Endogenous ACSVL3 was found primarily in mitochondria of MA-10 and Neuro2a cells by both Western blot analysis of subcellular fractions and immunofluorescence analysis. In MA-10 cells, loss-of-function studies using RNA interference confirmed that endogenous ACSVL3 is an acyl-CoA synthetase capable of activating both long-chain (C16:0) and very long-chain (C24:0) fatty acids. However, despite decreased acyl-CoA synthetase activity, initial rates of fatty acid uptake were unaffected by knockdown of Acsvl3 expression in MA-10 cells. These studies cast doubt on the designation of ACSVL3 as a fatty acid transport protein.

Acyl-CoA synthetases play a pivotal role in fatty acid metabolism, providing activated substrates for fatty acid catabolic and anabolic pathways. Acyl-CoA synthetases comprise numerous proteins with diverse substrate specificities, tissue expression patterns, and subcellular localizations, suggesting that each enzyme directs fatty acids toward a specific metabolic fate. We reported that hBG1, the human homolog of the acyl-CoA synthetase mutated in the Drosophila mutant “bubblegum,” belongs to a previously unidentified enzyme family and is capable of activating both long- and very long-chain fatty acid substrates. We now report that when overexpressed, hBG1 can activate diverse saturated, monosaturated, and polyunsaturated fatty acids. Using in situ hybridization and immunohistochemistry, we detected expression of mBG1, the mouse homolog of hBG1, in cerebral cortical and cerebellar neurons and in steroidogenic cells of the adrenal gland, testis, and ovary. The expression pattern and ability of BG1 to activate very long-chain fatty acids implicates this enzyme in the pathogenesis of X-linked adrenoleukodystrophy. In neuron-derived Neuro2a cells, mBG1 co-sedimented with mitochondria and was found in small vesicular structures located in close proximity to mitochondria. RNA interference was used to decrease mBG1 expression in Neuro2a cells and led to a 30-35% decrease in activation and β-oxidation of the long-chain fatty acid, palmitate. These results suggest that in Neuro2a cells, mBG1-activated long-chain fatty acids are directed toward mitochondrial degradation. mBG1 appears to play a minor role in very long-chain fatty acid activation in these cells, indicating that other acyl-CoA synthetases are necessary for very long-chain fatty acid metabolism in Neuro2a cells.

We report a case of a 71-year-old Caucasian male presenting with an exfoliative skin rash all over his body. The patient was also found to have a huge gall bladder mass extending into the liver that turned out to be adenocarcinoma of the gall bladder on biopsy. Gall bladder cancer usually presents with abdominal pain, swelling or jaundice. We report only the second case in literature of a gall bladder adenocarcinoma presenting with exfoliative erythroderma as its paraneoplastic presentation.

The association between the expression of excision repair cross‑complementing gene 1 (ERCC1), thymidylate synthase (TYMS), ribonuleotide reductase M1 (RRM1), βIII‑tubulin (TUBB3), non‑muscle myosin II, myoglobin and MyoD1 in metastatic lung adenocarcinoma, and clinical outcomes with platinum‑based chemotherapy treatment is not well‑established. Recently, increasing attention has been focused on the involvement of ERCC1, TYMS, RRM1 and TUBB3 in the development of drug resistance. There has been less research into the role of muscle myosin II, myoglobin and MyoD1 in the pathogenesis of lung cancer, although these genes are known to have important functions within tumor cells. In the current study, malignant pleural effusion from 116 patients with untreated lung adenocarcinoma diagnosed between 2011 and 2012, were collected. The protein expression levels of ERCC1, TYMS, RRM1 and TUBB3 were evaluated with immunocytochemistry and western blot analysis. The expression levels of non‑muscle myosin II, myoglobin and MyoD1 were measured in a subset of 50 patients, treated with platinum‑based chemotherapy. The association of each of these seven factors with one another, as well as with patient survival were analyzed. Immunohistochemistry demonstrated that the percentage of pleural fluid samples from patients with lung adenocarcinoma expressing ERCC1, TYMS, RRM1 and TUBB3 was 37, 36.2, 82.7 and 69.8%, respectively. In the subset of 50 patients in whom the remaining factors were analyzed, the percentage expressing non‑muscle myosin II was 48%, for myoglobin the figure was 40% and for MyoD1 it was 38%. There was a positive correlation between each pair of the above seven molecules with the exception of TYMS and RRM1. Expression of ERCC1, TYMS, TUBB3, non‑muscle myosin II, myoglobin and MyoD1 genes was associated with decreased survival in patients with metastatic lung adenocarcinoma. Expression of ERCC1, TYMS, TUBB3, non‑muscle myosin II, myoglobin and MyoD1 was also associated with decreased survival rates of patients with lung adenocarcinoma treated with platinum‑based chemotherapy. These factors may be used as clinical biomarkers to predict the biological behavior and chemoresistance of tumor cells, and the survival of patients with lung carcinoma.

3-Hydroxy-3-methylglutaryl-CoA lyase-like protein (HMGCLL1) has been annotated in the Mammalian Genome Collection as a previously unidentified human HMG-CoA lyase (HMGCL). To test the validity of this annotation and evaluate the physiological role of the protein, plasmids were constructed for protein expression in Escherichia coli and Pichia pastoris. Protein expression in E. coli produced insoluble material. In contrast, active HMGCLL1 could be recovered upon expression in P. pastoris. Antibodies were prepared against a unique peptide sequence found in the N terminus of the protein. In immunodetection experiments, the antibodies discriminated between HMGCLL1 and mitochondrial HMGCL. Purified enzyme was characterized and demonstrated to cleave HMG-CoA to acetoacetate and acetyl-CoA with catalytic and affinity properties comparable with human mitochondrial HMGCL. The deduced HMGCLL1 sequence contains an N-terminal myristoylation motif; the putative modification site was eliminated by construction of a G2A HMGCLL1. Modification of both proteins was attempted using human N-myristoyltransferase and [(3)H]myristoyl-CoA. Wild-type protein was clearly modified, whereas G2A protein was not labeled. Myristoylation of HMGCLL1 affects its cellular localization. Upon transfection of appropriate expression plasmids into COS1 cells, immunofluorescence detection indicates that G2A HMGCLL1 exhibits a diffuse pattern, suggesting a cytosolic location. In contrast, wild-type HMGCLL1 exhibits a punctate as well as a perinuclear immunostaining pattern, indicating myristoylation dependent association with nonmitochondrial membrane compartments. In control experiments with the HMGCL expression plasmid, protein is localized in the mitochondria, as anticipated. The available results for COS1 cell expression, as well as endogenous expression in U87 cells, indicate that HMGCLL1 is an extramitochondrial hydroxymethylglutaryl-CoA lyase.

Expression of hydroxymethylglutaryl-CoA lyase like protein (HMGCLL1) in Pichia pastoris produces the 340 residue human protein. Isolated protein catalyzes ketogenic HMG-CoA cleavage with efficiency...

The contribution of lipid metabolic pathways to malignancy is poorly understood. Expression of the fatty acyl-CoA synthetase ACSVL3 was found to be markedly elevated in clinical malignant glioma specimens but nearly undetectable in normal glia. ACSVL3 levels correlated with the malignant behavior of human glioma cell lines and glioma cells propagated as xenografts. ACSVL3 expression was induced by the activation of oncogenic receptor tyrosine kinases (RTK) c-Met and epidermal growth factor receptor. Inhibiting c-Met activation with neutralizing anti–hepatocyte growth factor monoclonal antibodies reduced ACSVL3 expression concurrent with tumor growth inhibition in vivo. ACSVL3 expression knockdown using RNA interference, which decreased long-chain fatty acid activation, inhibited anchorage-dependent and anchorage-independent glioma cell growth by ∼70% and ∼90%, respectively. ACSVL3-depleted cells were less tumorigenic than control cells, and subcutaneous xenografts grew ∼60% slower than control tumors. Orthotopic xenografts produced by ACSVL3-depleted cells were 82% to 86% smaller than control xenografts. ACSVL3 knockdown disrupted Akt function as evidenced by RTK-induced transient decreases in total and phosphorylated Akt, as well as glycogen synthase kinase 3β, via a caspase-dependent mechanism. Expressing constitutively active myr-Akt rescued cells from the anchorage-dependent and anchorage-independent growth inhibitory effects of ACSVL3 depletion. These studies show that ACSVL3 maintains oncogenic properties of malignant glioma cells via a mechanism that involves, in part, the regulation of Akt function. [Cancer Res 2009;69(24):9175–82]

Background: The dopamine (DA) D2 receptor (D2R) agonist bromocriptine (BC) decreases body fat in animal and human models and increases lean muscle mass, improves glucose intolerance and insulin resistance, and reduces triglycerides and free fatty acids. We have previously shown a negative correlation between D2R and body weight in obese individuals and in rodents, and that chronic food restriction increases D2R binding in genetically obese rats. The purpose of this study was to assess whether the antiobesity and metabolic effects of BC are related to changes in midbrain DA and D2R activity by measuring D2R and DA transporter (DAT) binding in a genetic (leptin-receptor-deficient) and environmental (diet-induced) rodent obesity model. Methods: Obese (fa/fa) (leptin-receptor-deficient), lean (FA/FA) Zucker rats and rats with diet-induced obesity (DIO) were treated with 10 mg/kg BC for 4 weeks. Body weight, food intake, locomotor activity and blood glucose levels were measured along with D2R- and DAT-binding levels using in vitro receptor autoradiography. Results: BC decreased food intake and body fat and increased locomotor activity in both the (fa/fa) and DIO rats. Furthermore, BC increased D2R binding in (fa/fa) but not in DIO rats. Finally, BC increased DAT binding in DIO rats but not in the (fa/fa) rats. Conclusion: These observations are all consistent with the existence of unique leptin-DA interactions and the hypothesis that there is hyposensitivity of the DA system in obesity.

Fatty acid transport protein 4 (FATP4) is a fatty acyl-CoA synthetase that preferentially activates very long chain fatty acid substrates, such as C24:0, to their CoA derivatives. To gain better insight into the physiological functions of FATP4, we established dermal fibroblast cell lines from FATP4-deficient wrinkle-free mice and wild type (w.t.) mice. FATP4 -/- fibroblasts had no detectable FATP4 protein by Western blot. Compared with w.t. fibroblasts, cells lacking FATP4 had an 83% decrease in C24:0 activation. Peroxisomal degradation of C24:0 was reduced by 58%, and rates of C24:0 incorporation into major phospholipid species (54–64% decrease), triacylglycerol (64% decrease), and cholesterol esters (58% decrease) were significantly diminished. Because these lipid metabolic processes take place in different subcellular organelles, we used immunofluorescence and Western blotting of subcellular fractions to investigate the distribution of FATP4 protein and measured enzyme activity in fractions from w.t. and FATP4 -/- fibroblasts. FATP4 protein and acyl-CoA synthetase activity localized to multiple organelles, including mitochondria, peroxisomes, endoplasmic reticulum, and the mitochondria-associated membrane fraction. We conclude that in murine skin fibroblasts, FATP4 is the major enzyme producing very long chain fatty acid-CoA for lipid metabolic pathways. Although FATP4 deficiency primarily affected very long chain fatty acid metabolism, mutant fibroblasts also showed reduced uptake of a fluorescent long chain fatty acid and reduced levels of long chain polyunsaturated fatty acids. FATP4-deficient cells also contained abnormal neutral lipid droplets. These additional defects indicate that metabolic abnormalities in these cells are not limited to very long chain fatty acids.

Acyl-CoA synthetases that activate fatty acids to their CoA derivatives play a central role in fatty acid metabolism. ACSBG1, an acyl-CoA synthetase originally identified in the fruit fly mutant bubblegum, was hypothesized to contribute to the biochemical pathology of X-linked adrenoleukodystrophy. We looked for homologous proteins and identified ACSBG2 in humans, mice, and rats. Human ACSBG1 and ACSBG2 amino acid sequences are 50% identical. ACSBG2 expression was confined to the testis and brainstem. Immunohistochemistry and in situ hybridization studies further localized ACSBG2 expression to testicular Sertoli cells and large motoneurons in the medulla oblongata and cervical spinal cord. Full-length cDNA encoding human and mouse ACSBG2 was cloned. In transfected COS-1 cells, both human and murine ACSBG2 were detected as 75- to 80-kDa proteins by Western blot. Cells overexpressing ACSBG2 had increased ability to activate oleic acid (C18:1omega9) and linoleic acid (C18:2omega6) but not other fatty acid substrates tested. Within a highly conserved motif known to be important for catalysis, human ACSBG2 contains a histidine residue where all other known acyl-CoA synthetases, including mouse and rat ACSBG2, contain an arginine. This substitution resulted in a shift of the human ACSBG2 pH optimum to a more acidic pH. Mutation of this histidine to arginine improved catalytic function at neutral pH by shifting the pH profile without affecting substrate specificity. Although the role of ACSBG2 in testicular and neuronal lipid metabolism remains unclear, the limited tissue expression pattern and limited substrate specificity rule out a likely role for this enzyme in X-linked adrenoleukodystrophy pathology.

The principal biochemical abnormality in the neurodegenerative disorder X-linked adrenoleukodystrophy (X-ALD) is elevated plasma and tissue levels of very long-chain fatty acids (VLCFA). Enzymes with very long-chain acyl-CoA synthetase (VLACS) activity are required for VLCFA metabolism, including degradation by peroxisomal beta-oxidation or incorporation into complex lipids, and may also participate in VLCFA synthesis. Two enzymes with VLACS activity, ACSVL1 and BG1, were investigated for their potential role in X-ALD biochemical pathology. Skin fibroblast mRNA levels for ACSVL1, an enzyme previously shown to be in peroxisomes and to participate in VLCFA beta-oxidation, were not significantly different between normal controls, patients with childhood cerebral X-ALD, and patients with adrenomyeloneuropathy. Similar results were obtained with mRNA for BG1, a non-peroxisomal enzyme that is highly expressed in nervous system, adrenal gland, and testis, the principal tissues pathologically affected in X-ALD. No significant differences in the immunohistochemical staining patterns of tissues expressing either ACSVL1 or BG1 were observed when wild-type and X-ALD mice were compared. Western blot analysis of BG1 protein levels showed no differences between fibroblasts from controls, cerebral X-ALD, or adrenomyeloneuropathy patients. BG1 protein levels were similar in wild-type and X-ALD mouse brain, spinal cord, testis, and adrenal gland. We hypothesized that one function of BG1 was to direct VLCFA into the cholesterol ester synthesis pathway. However, BG1 depletion in Neuro2a cells using RNA interference did not decrease incorporation of labeled VLCFA into cholesterol esters. We conclude that the role, if any, of ACSVL1 and BG1 in X-ALD biochemical pathology is indirect.

Acyl-CoA synthetases play a pivotal role in fatty acid metabolism, providing activated substrates for fatty acid catabolic and anabolic pathways. Acyl-CoA synthetases comprise numerous proteins with diverse substrate specificities, tissue expression patterns, and subcellular localizations, suggesting that each enzyme directs fatty acids toward a specific metabolic fate. We reported that hBG1, the human homolog of the acyl-CoA synthetase mutated in the Drosophila mutant "bubblegum," belongs to a previously unidentified enzyme family and is capable of activating both long- and very long-chain fatty acid substrates. We now report that when overexpressed, hBG1 can activate diverse saturated, monosaturated, and polyunsaturated fatty acids. Using in situ hybridization and immunohistochemistry, we detected expression of mBG1, the mouse homolog of hBG1, in cerebral cortical and cerebellar neurons and in steroidogenic cells of the adrenal gland, testis, and ovary. The expression pattern and ability of BG1 to activate very long-chain fatty acids implicates this enzyme in the pathogenesis of X-linked adrenoleukodystrophy. In neuron-derived Neuro2a cells, mBG1 co-sedimented with mitochondria and was found in small vesicular structures located in close proximity to mitochondria. RNA interference was used to decrease mBG1 expression in Neuro2a cells and led to a 30-35% decrease in activation and beta-oxidation of the long-chain fatty acid, palmitate. These results suggest that in Neuro2a cells, mBG1-activated long-chain fatty acids are directed toward mitochondrial degradation. mBG1 appears to play a minor role in very long-chain fatty acid activation in these cells, indicating that other acyl-CoA synthetases are necessary for very long-chain fatty acid metabolism in Neuro2a cells.

Bile acids are synthesized de novo in the liver from cholesterol and conjugated to glycine or taurine via a complex series of reactions involving multiple organelles. Bile acids secreted into the small intestine are efficiently reabsorbed and reutilized. Activation by thioesterification to CoA is required at two points in bile acid metabolism. First, 3alpha,7alpha,12alpha-trihydroxy-5beta-cholestanoic acid, the 27-carbon precursor of cholic acid, must be activated to its CoA derivative before side chain cleavage via peroxisomal beta-oxidation. Second, reutilization of cholate and other C24 bile acids requires reactivation prior to re-conjugation. We reported previously that homolog 2 of very long-chain acyl-CoA synthetase (VLCS) can activate cholate (Steinberg, S. J., Mihalik, S. J., Kim, D. G., Cuebas, D. A., and Watkins, P. A. (2000) J. Biol. Chem. 275, 15605-15608). We now show that this enzyme also activates chenodeoxycholate, the secondary bile acids deoxycholate and lithocholate, and 3alpha,7alpha,12alpha-trihydroxy-5beta-cholestanoic acid. In contrast, VLCS activated 3alpha,7alpha,12alpha-trihydroxy-5beta-cholestanoate, but did not utilize any of the C24 bile acids as substrates. We hypothesize that the primary function of homolog 2 is in the reactivation and recycling of C24 bile acids, whereas VLCS participates in the de novo synthesis pathway. Results of in situ hybridization, topographic orientation, and inhibition studies are consistent with the proposed roles of these enzymes in bile acid metabolism.

Cellular fatty acids typically derive from uptake from the extracellular milieu and, to a lesser extent, de novo synthesis. Extracellular fatty acids must traverse the plasma membrane, after which they are activated to their CoA thioesters for subsequent metabolism. Both uptake and metabolism are rapid processes, and there has been considerable debate as to whether transport of fatty acids across the lipid bilayer of the plasma membrane proceeds by diffusion or requires transport proteins. One group of proteins proposed to translocate fatty acids is the six-member Fatty Acid Transport Protein (FATP) family. These proteins were designated as such because when overexpressed, host cells exhibited higher rates of accretion of radioactive or fluorescent fatty acids. However, one member of this family, FATP2, is identical to an enzyme with very long-chain acyl-CoA synthetase (ACSVL) activity. This enzyme (ACSVL1 or FATP2), was isolated using classical protein purification techniques. In fact, the six-member ACSVL protein family is identical to the six-member FATP family. We and others have established that all six proteins have acyl-CoA synthetase activity. It remains to be established whether they participate in the physical translocation process, or facilitate transport by trapping, as CoA derivatives, fatty acids that enter cells by diffusion. To characterize the biological functions of the ACSVLs, we are investigating the properties of the overexpressed proteins and the endogenous proteins. We observed that for many ACSVLs, the subcellular location of the overexpressed protein differs from that of the endogenous protein. Using RNA interference (siRNA), we knocked down expression of FATP4 (proposed name: ACSVL5) in Neuro2a cells. Activation of both long-chain (C16:0) and very long-chain fatty acids (C24:0) was decreased when FATP4 was depleted. Despite decreased enzyme activity, initial rates of uptake of [14C]C16:0 were not affected when FATP4 was depleted. In contrast, COS-1 cells overexpressing FATP4 showed enhanced [14C]C16:0 uptake. Neither endogenous (Neuro2a) nor overexpressed (COS-1) FATP4 was localized to plasma membrane under routine cell culture conditions, but rather were found in intracellular membrane compartments. We conclude that, in the cell lines studied, endogenous FATP4 does not function to translocate FA across the plasma membrane.