Counter modulation of adipocyte mitochondrial processes by insulin and S-oxalylglutathione.

Oxalyl thiolesters, a group of putative intracellular regulators, have been shown to be in vitro inhibitors of some cytosolic enzymes which are stimulated by insulin. In this study, the effects of insulin and oxalyl thiolesters on pyruvate dehydrogenase, beta-oxidation, and acyl-CoA hydrolase activities in mitochondria from rat epididymal adipocytes are compared. Using glutathione, CoASH, cysteine, and cysteamine as thiol sources, oxalyl thioesters were synthesized, purified, and quantitated. Mitochondria were isolated from rat epididymal adipocytes, some of which were incubated with or without insulin. Mitochondrial activities were determined by radioisotopic assay subsequent to control, insulin, or oxalyl thiolester incubation. Under the conditions used in this study, pyruvate dehydrogenase activity was increased 28% subsequent to 10-min incubation of adipocytes with 400 microU/ml insulin; in contrast, preincubation of adipocyte mitochondria with S-oxalylglutathione resulted in a dose-dependent 11-19% inhibition of pyruvate dehydrogenase. S-oxalylglutathione also attenuated the spermine-induced activation of pyruvate dehydrogenase. Insulin treatment resulted in a small but significant increase in beta-oxidation of palmitic acid while 100 microM S-oxalylglutathione mediated a 40% decrease in palmitate oxidation. Palmitoyl-CoA hydrolase activity was decreased 14% by insulin treatment; however, S-oxalylglutathione caused a 14-50% increase in hydrolase activity. The other oxalyl thiolesters were not as effective or as consistent as S-oxalylglutathione in modulation of the mitochondrial activities; free thiols and oxalic acid did not modulate the activities. In summary, pyruvate dehydrogenase, palmitate beta-oxidation, and palmitoyl-CoA hydrolase activities in adipocyte mitochondria were modulated in approximately equal but opposite directions by insulin and S-oxalylglutathione. These findings support the suggestion that oxalyl thiolesters may function as an intracellular signal recruited to return insulin to normal levels.

Species differences in the intracellular distribution of ciprofibroyl-CoA hydrolase. Implications for peroxisome proliferation.

Peroxisomal proliferators (HPP), such as ciprofibrate and clofibric acid, are species-specific drugs. Since HPP-coenzyme A derivatives might be involved in their action, we studied the subcellular distribution of liver ciprofibroyl-CoA hydrolase in rat and in two HPP-unresponsive species, humans and guinea pig. Total activity was similar in the three species and was not induced by clofibric acid treatment. In guinea pig, as in humans, the enzyme is localized in the mitochondrial and soluble fractions and no changes are observed after drug treatment. In the rat, the enzyme has a microsomal localization, but upon clofibric acid treatment it changes to a mitochondrial and soluble distribution, as in unresponsive species. These results raise the possibility that drug-induced hydrolases in rats might be normally expressed in humans and guinea pigs.

Very-long-chain fatty acids activate lysosomal hydrolases in neonatal human skin tissue.

OBJECTIVE: The aim of this study was to examine the in vitro effect of peroxisomal dysfunction on lysosomal enzymes, the autophagic machinery in the cell, in order to understand the mechanisms of pathogenesis of peroxisomal disorders. MATERIALS AND METHODS: Foreskin samples were obtained immediately after circumcision of 1- to 2-day-old infants at the Maternity Hospital, Kuwait. Skin tissues were cleaned, cut into slices of 1-2 mm2 in size and treated with lignoceric acid (1-20 microg/ml), a very-long-chain fatty acid (VLCFA), in the presence or absence of 1-5 mM aminotriazole (ATZ). A battery of lysosomal enzymes were assayed following treatment of dermal tissue with VLCFA or ATZ. RESULTS: Treatment of skin slices with lignoceric acid significantly increased (p < 0.001) the enzymic activities of acid lipase, acid phosphatase, alpha-glucosidase, alpha-galactosidase, N-acetyl-alpha-D-glucosaminidase (NAGA) and N-acetyl-alpha-D-galactosaminidase (NAGTA). ATZ (1-5 mM), an inhibitor of key peroxi somal enzyme catalase, also markedly increased the enzymic activities of acid phosphatase, alpha-glucosidase (23%) and alpha-galactosidase (18%) without any significant effect on NAGA or NAGTA. Western blot analysis further revealed that both VLCFA and ATZ significantly increased the protein expression of lysosomal enzymes, beta-galactosidase and beta-glucuronidase. CONCLUSION: Experimen tal dysfunction of peroxisomes mimicked by elevated VLCFA or ATZ-mediated catalase inhibition significantly increased the activities of lysosomal hydrolases in human dermal tissue, suggesting that activation of the lysosomal system could be one of the factors responsible for cellular damage during pathogenesis of peroxisomal diseases.

Purification, characterization and modulation of a microsomal carboxylesterase in rat liver for the hydrolysis of acyl-CoA.

A carboxylesterase containing long-chain acyl-CoA hydrolase activity was purified to apparent homogeneity from rat liver microsomes. Palmitoyl-CoA was the most preferred substrate, followed by stearoyl-CoA and oleoyl-CoA. Arachidonoyl-CoA, linoleoyl-CoA and acetyl-CoA were not hydrolysed by the enzyme. The purified enzyme had no activity on the hydrolysis of phospholipids and neutral lipids. The molecular mass of the enzyme was found to be 56 kDa by SDS/PAGE and 64 kDa by gel-filtration chromatography. On isoelectric focusing, the purified enzyme behaved like the ES-4 type, with a pI of 6.15. Determination of the amino acid sequence revealed that its N-terminal sequence is 100% homologous with the only other known N-terminal sequence for a rat carboxylesterase isoenzyme (ES-10). Enzyme activity was inhibited by lysophosphatidic acid and activated by lysophosphatidylcholine. The modulation of enzyme activity by these lysophospholipids might represent a plausible mechanism for the physiological control of acyl-CoA concentrations.

Peroxisome proliferators differentially regulate long-chain acyl-CoA thioesterases in rat liver.

We have investigated the effects of peroxisome proliferators on rat liver long-chain acyl-CoA thioesterase activities. Subcellular fractionations of liver homogenates from control, clofibrate- and di(2-ethylhexyl)phthalate-treated rats confirmed earlier studies which demonstrated that peroxisome-proliferating drugs induce long-chain acyl-CoA thioesterase activity mainly in the mitochondrial and cytosolic fractions. The aim of the present study was to investigate whether the induced activities were due to increases in normally expressed enzymes, or due to induction of novel enzymes. To investigate whether structurally different peroxisome proliferators differentially induced thioesterase activities, we tested the effects of di(2-ethylhexyl)phthalate (a plastisizer) and the hypolipidemic drug clofibrate. For this purpose, we established an analytical size exclusion chromatography method. Chromatography of solubilised mitochondrial matrix proteins showed that the activity in control mitochondria was mainly due to enzymes with molecular masses of about 50 kDa and 35 kDa. The activity in samples prepared from clofibrate- and di(2-ethylhexyl)phthalate-treated rats eluted as proteins of about 40 kDa and 110 kDa. Highly purified peroxisomes contained two peaks of activity, which were not induced, that corresponded to molecular masses of 40 kDa and 80 kDa. The 80-kDa peak was shown to be due to dimerization by addition of glycerol. Chromatography of cytosolic fractions from control rat livers indicated the presence of long-chain acyl-CoA thioesterases with molecular masses of approximately 35 kDa and 125 kDa and a broad peak corresponding to a high-molecular-mass protein. The activity in cytosolic fractions from peroxisome-proliferator-treated rats eluted mainly as peaks corresponding to 40, 110 and 150 kDa. In addition, in the 110-kDa peak, a different degree of induction and different chain-length specificities were caused by clofibrate and di(2-ethylhexyl)phthalate, suggesting that these peroxisome proliferators differentially regulate the cytosolic acyl-CoA thioesterase activities. Western blot analysis showed that enzymes in the 40-kDa peak of the peroxisomal and cytosolic fractions were structurally related, but not identical, to a 40-kDa mitochondrial very-long-chain acyl-CoA thioesterase. Our data show that the increased acyl-CoA thioesterase activities in mitochondria and cytosol were mainly due to induction of acyl-CoA thioesterases which are not, or only weakly, expressed under normal conditions.

Structural basis for the insensitivity of a serine enzyme (palmitoyl-protein thioesterase) to phenylmethylsulfonyl fluoride.

Palmitoyl-protein thioesterase-1 (PPT1) is a newly described lysosomal enzyme that hydrolyzes long chain fatty acids from lipid-modified cysteine residues in proteins. Deficiency in this enzyme results in a severe neurodegenerative storage disorder, infantile neuronal ceroid lipofuscinosis. Although the primary structure of PPT1 contains a serine lipase consensus sequence, the enzyme is insensitive to commonly used serine-modifying reagents phenylmethylsulfonyl fluoride (PMSF) and diisopropylfluorophosphate. In the current paper, we show that the active site serine in PPT1 is modified by a substrate analog of PMSF, hexadecylsulfonylfluoride (HDSF) in a specific and site-directed manner. The apparent K(i) of the inhibition was 125 micrometer (in the presence of 1.5 mm Triton X-100), and the catalytic rate constant for sulfonylation (k(2)) was 3.3/min, a value similar to previously described sulfonylation reactions. PPT1 was crystallized after inactivation with HDSF, and the structure of the inactive form was determined to 2.4 A resolution. The hexadecylsulfonyl was found to modify serine 115 and to snake through a narrow hydrophobic channel that would not accommodate an aromatic sulfonyl fluoride. Therefore, the geometry of the active site accounts for the reactivity of PPT1 with HDSF but not PMSF. These observations suggest a structural explanation as to why certain serine lipases are resistant to modification by commonly used serine-modifying reagents.

Antisense palmitoyl protein thioesterase 1 (PPT1) treatment inhibits PPT1 activity and increases cell death in LA-N-5 neuroblastoma cells.

Infantile neuronal ceroid lipofuscinosis (INCL) is a childhood neurodegenerative disease caused by the selective death of cortical neurons and retinal degeneration, as the result of a palmitoyl protein thioesterase 1 (PPT1) deficiency. Recently, we showed that overexpression of PPT1 protects LA-N-5 human neuroblastoma cells against apoptotic death (Cho and Dawson [2000a] J. Neurochem. 74:1478-1488) and we now show that inhibition of PPT1 increases the susceptibility of these cells to apoptotic cell death. Transient transfection of LA-N-5 neuroblastoma cells with PPT1-FLAG resulted in a strong expression of PPT-FLAG-tagged protein as evidenced by Western blot analysis and immunofluorescence. Co-transfection of a reverse-oriented (antisense) PPT1 (AS-PPT1) decreased the expression of PPT-FLAG to almost zero, reduced PPT1 enzyme activity (as measured by an in vitro assay) and increased the susceptibility to apoptosis induced by C(2) ceramide. Similarly, inhibition of PPT1 with a synthetic inhibitor (AcG-palmitoyl diaminoproprionate-VKIKK) (DAP1) (100 microM) increased the susceptibility of the cells to apoptosis induced by either C(2)-ceramide or etoposide, a common chemotherapeutic agent used in the treatment of neuroblastoma. Cells stably overexpressing PPT1 were resistant to apoptosis induced by DAP1 suggesting that the inhibitor has a specific action and confirming that low levels of protein palmitoylation block the death pathway. Drugs that raise the level of protein palmitoylation are pro-apoptotic and PPT1 inhibition may enhance the killing efficacy of chemotherapeutic agents used to kill neuroblastoma-derived cells.

Characterization of Aquamicrobium defluvii gen. nov. sp. nov., a thiophene-2-carboxylate-metabolizing bacterium from activated sludge.

A gram-negative bacterium was isolated from activated sewage sludge with thiophene-2-carboxylate as the sole source of carbon and with nitrate as an electron acceptor. The isolate, strain NKK, was a motile, oxidase- and catalase-positive, rod-like bacterium with a G+C content of 61.7 mol%. Besides nitrate, oxygen could serve as a terminal electron acceptor. Among many carbon sources tested, only a few sugars, fatty acids, and thiophene-2-carboxylate supported growth. Other heterocyclic compounds were not used. The sulfur atom of thiophene-2-carboxylate was oxidized to thiosulfate when cells were grown aerobically, or to elemental sulfur when cells were grown anaerobically with nitrate. Nitrate was reduced to nitrite. Growth on thiophene-2-carboxylate was dependent on the addition of molybdate to the medium. Tungstate, a specific antagonist of molybdate, inhibited growth on thiophene-2-carboxylate at concentrations > 10(-7) M. Three inducible enzymes involved in the metabolism of thiophene-2-carboxylate were detected: an ATP-, CoA-, thiophene-2-carboxylate- and Mg2+-dependent thiophene-2-carboxyl-CoA ligase (AMP-forming), a molybdenum-containing thiophene-2-carboxyl-CoA dehydrogenase, and a thiophene-2-carboxyl-CoA thioesterase. The sequence of the 16S rRNA gene suggested a classification of strain NKK within the alpha-subgroup of the Proteobacteria as a new genus and species, Aquamicrobium defluvii gen. nov. sp. nov. (DSM 11603), closely related to Mesorhizobium sp. and Phyllobacterium sp., but representing a distinct lineage equal in depth to those of the two mentioned genera. Aquamicrobium defluvii can be distinguished from both genera by a distinct spectrum of substrates, the maximal growth temperature, and a different salt tolerance.

Evidence for mitochondrial thioesterase 1 as a peroxisome proliferator-activated receptor-alpha-regulated gene in cardiac and skeletal muscle.

The physiological role of mitochondrial thioesterase 1 (MTE1) is unknown. It was proposed that MTE1 promotes fatty acid (FA) oxidation (FAO) by acting in concert with uncoupling protein (UCP)3. We previously showed that ucp3 is a peroxisome proliferator-activated receptor-alpha (PPAR alpha)-regulated gene, allowing induction when FA availability increases. On the assumption that UCP3 and MTE1 act in partnership to increase FAO, we hypothesized that mte1 is also a PPAR alpha-regulated gene in cardiac and skeletal muscle. Using real-time RT-PCR, we characterized mte1 gene expression in rat heart and soleus muscles. Messenger RNA encoding for mte1 was 3.2-fold higher in heart than in soleus muscle. Cardiac mte1 mRNA exhibited modest diurnal variation, with 1.4-fold higher levels during dark phase. In contrast, skeletal muscle mte1 mRNA remained relatively constant over the course of the day. High-fat feeding, fasting, and streptozotocin-induced diabetes, interventions that increase FA availability, muscle PPAR alpha activity, and muscle FAO rates, increased mte1 mRNA in heart and soleus muscle. Conversely, pressure overload and hypoxia, interventions that decrease cardiac PPAR alpha activity and FAO rates, repressed cardiac mte1 expression. Specific activation of PPAR alpha in vivo through WY-14643 administration rapidly induced mte1 mRNA in cardiac and skeletal muscle. WY-14643 also induced mte1 mRNA in isolated adult rat cardiomyocytes dose dependently. Expression of mte1 was markedly lower in hearts and soleus muscles isolated from PPAR alpha-null mice. Alterations in cardiac and skeletal muscle ucp3 expression mirrored that of mte1 in all models investigated. In conclusion, mte1, like ucp3, is a PPAR alpha-regulated gene in cardiac and skeletal muscle.