Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.3.1.21 (CPT)
4,580 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Acidification of the endosomal pathway is important for ligand and receptor sorting, toxin activation, and protein degradation by lysosomal acid hydrolases. Fluorescent probes and imaging methods were developed to measure pH to better than 0.2 U accuracy in individual endocytic vesicles in Swiss 3T3 fibroblasts. Endosomes were pulse labeled with transferrin (Tf), alpha 2-macroglobulin (alpha 2M), or dextran, each conjugated with tetramethylrhodamine and carboxyfluorescein (for pH 5-8) or dichlorocarboxyfluorescein (for pH 4-6); pH in individual labeled vesicles was measured by ratio imaging using a cooled CCD camera and novel image analysis software. Tf-labeled endosomes acidified to pH 6.2 +/- 0.1 with a t1/2 of 4 min at 37 degrees C, and remained small and near the cell periphery. Dextran- and alpha 2M-labeled endosomes acidified to pH 4.7 +/- 0.2, becoming larger and moving toward the nucleus over 30 min; approximately 15% of alpha 2M-labeled endosomes were strongly acidic (pH less than 5.5) at only 1 min after labeling. Replacement of external Cl by NO3 or isethionate strongly and reversibly inhibited acidification. Addition of ouabain (1 mM) at the time of labeling strongly enhanced acidification in the first 5 min; Tf-labeled endosomes acidified to pH 5.3 without a change in morphology. Activation of phospholipase C by vasopressin (50 nM) enhanced acidification of early endosomes; activation of protein kinase C by PMA (100 nM) enhanced acidification strongly, whereas elevation of intracellular Ca by A23187 (1 microM) had no effect on acidification. Activation of protein kinase A by CPT-cAMP (0.5 mM) or forskolin (50 microM) inhibited acidification. Lysosomal pH was not affected by ouabain or the protein kinase activators. These results establish a methodology for quantitative measurement of pH in individual endocytic vesicles, and demonstrate that acidification of endosomes labeled with Tf and alpha 2M (receptor-mediated endocytosis) and dextran (fluid-phase endocytosis) is sensitive to intracellular anion composition, Na/K pump inhibition, and multiple intracellular second messengers.
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PMID:Second messengers regulate endosomal acidification in Swiss 3T3 fibroblasts. 138 79

An assay procedure for carnitine palmitoyltransferase is described which allows rapid measurement of the overt activity of this enzyme in isolated rat hepatocytes. In a one-step procedure digitonin permeabilizes the plasma membrane and at the same time carnitine palmitoyltransferase activity is measured. The use of the present procedure shows that carnitine palmitoyltransferase activity is regulated on the short term by different types of agonists. Thus, insulin, epidermal growth factor, vasopressin and the phorbol ester PMA inhibit carnitine palmitoyltransferase activity, whereas glucagon treatment renders the enzyme more active. These changes in enzyme activity coincide with corresponding changes in the rate of fatty acid oxidation.
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PMID:Short-term regulation of carnitine palmitoyltransferase activity in isolated rat hepatocytes. 334 11

In PC12 cells, cAMP stimulates the MAP kinase pathway by an unknown mechanism. Firstly, we examined the role of calcium ion mobilization and of protein kinase C in cAMP-stimulated MAP kinase activation. We show that cAMP stimulates p44mapk independently of these events. Secondly, we studied the role of B-Raf in this process. We observed that NGF, PMA and cAMP induce the phosphorylation of B-Raf as well as an upward shift in its electrophoretic mobility. We show that B-Raf is activated following NGF and PMA treatment of PC12 cells, and that it can phosphorylate and activate MEK-1. However, cAMP inhibits B-Raf autokinase activity as well as its ability to phosphorylate and activate MEK-1. This inhibition is likely to be due to a direct effect since we found that PKA phosphorylates B-Raf in vitro. Further, we show that B-Raf binds to p21ras, but more important, this binding to p21ras is virtually abolished with B-Raf from PC12 cells treated with CPT-cAMP. Hence, these data indicate that the PKA-mediated phosphorylation of B-Raf hampers its interaction with p21ras, which is responsible for the PKA-mediated decrease in B-Raf activity. Finally, our work suggests that in PC12 cells, cAMP stimulates MAP kinase through the activation of an unidentified MEK kinase and/or the inhibition of a MEK phosphatase.
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PMID:Regulation of the MAP kinase cascade in PC12 cells: B-Raf activates MEK-1 (MAP kinase or ERK kinase) and is inhibited by cAMP. 783 30

The aim was to investigate if chronic feeding with oligofructose (OFS), a nondigestible fructan that decreases triacylglycerol-very-low-density lipoproteins (TAG-VLDLs) in the serum of rats by reducing hepatic de novo lipogenesis, could counteract the impact of fructose on TAG metabolism. Male Wistar rats fed a standard diet supplemented or not with 10% OFS for 30 days received either tap water or a 10% fructose drinking solution for 48 hours. TAG, phospholipids (PLs), cholesterol, and free fatty acids were assayed both in serum and in liver. Fatty acid de novo synthesis, esterification, and beta-oxidation were assessed in the liver by measuring the activity of key enzymes: fatty acid synthase (FAS), phosphatidate phosphohydrolase (PAP), glycerol-3-phosphate acyltransferase (GPAT), and carnitine palmitoyltransferase-I (CPT-I), respectively. The acute load of fructose increased (1) both liver and serum TAG without affecting other lipids, and (2) de novo fatty acid synthesis and esterification, through induction of FAS and PAP without affecting CPT-I. Long-term feeding with OFS protected rats against liver TAG accumulation induced by fructose. The lower lipogenic capacity of the liver could be the key event in this protection, since even after the fructose load FAS activity remained significantly lower in OFS-fed rats. However, despite its protective effect on the liver, OFS was not able to prevent fructose-induced hypertriglyceridemia, suggesting that OFS feeding could not counteract the fructose-induced defect in TAG-VLDL clearance.
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PMID:Dietary oligofructose modifies the impact of fructose on hepatic triacylglycerol metabolism. 896 90

Previously we reported that phorbol ester, a protein kinase C (PKC) activator, exhibits a unique pattern of potentiation of nitric oxide (NO)-related apoptosis in HL-60 human promyelocytic leukemia cells. Here we show that elevation of intracellular cAMP could protect HL-60 cells from NO- or NO plus PMA-induced DNA damage. Exposure of cells to sodium nitroprusside (SNP; 0.5 to 4 mM), a NO-generating agent, induced apoptotic cell death as monitored by morphological means, gel electrophoresis, and in situ TdT-apoptosis assay. However, concomitant incubation of the cells with DB-cAMP markedly inhibited SNP-induced apoptotic cell death in a dose-dependent manner. Similar results were obtained with other commonly used cAMP analogs such as CPT-cAMP and 8-C1-cAMP and the intracellular cAMP-elevating agent such as forskolin. In contrast, pretreatment of HL-60 cells with H89 or KT5720, which are known to inhibit cAMP-dependent protein kinase (PKA), abolished the protective effect of cAMP analogs and forskolin on SNP-induced apoptosis. Synergism between SNP and phorbol ester to induce apoptosis was also inhibited by prior treatment of HL-60 cells with DB-cAMP or forskolin. The effect of DB-cAMP in maintaining cell viability was not associated with the onset of G0/G1 cell cycle arrest. In addition, neither dimethyl sulfoxide nor retinoic acid (which produce granulocyte differentiation) could produce cAMP effect. Under the same conditions, DB-cAMP also inhibited NO- or NO plus phorbol ester-induced apoptosis in another transformed cell line, U-937 cells. Taken together, these findings suggest that exposure of HL-60 cells to cAMP analogs renders them more resistant to NO-induced DNA damage and further suggest the existence of specific down-modulatory mechanisms related to NO-induced apoptotic DNA fragmentation.
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PMID:Cyclic adenosine monophosphate inhibits nitric oxide-induced apoptosis in human leukemic HL-60 cells. 957 15

Agents that increase intracellular cAMP have been shown to reduce joint inflammation in experimental arthritis, presumably by lowering the release of proinflammatory cytokines, such as TNF-alpha. Recent studies suggest that, in joints of patients with rheumatoid arthritis, TNF-alpha release from macrophages is triggered by their interaction with IL-15-stimulated T lymphocytes. In this report, we analyze the effect of rolipram, a cAMP-specific phosphodiesterase inhibitor, on TNF-alpha production in this experimental system. Cocultures of U937 cells with IL-15-stimulated T cells, but not control T cells, resulted in increased release of TNF-alpha. Pretreatment of T cells with rolipram or cAMP analogues inhibited the IL-15-stimulated increases in proliferation, expression of cell surface molecules CD69, ICAM-1, and LFA-1, and release of TNF-alpha from macrophages. Addition of PMA to T cells dramatically increased the expression of cell surface molecules, but had little or no effect on TNF-alpha release from either T cells or from cocultures, suggesting that other surface molecules must also be involved in T cell/macrophage contact-mediated production of TNF-alpha. Addition of PMA synergistically increased the proliferation of IL-15-stimulated T cells and the secretion of TNF-alpha from IL-15-stimulated T cell/macrophage cocultures. Rolipram and 8-(4-chlorophenylthio)-cAMP (CPT-cAMP) blocked these increases. Measurement of protein kinase A (PKA) activity and the use of inhibitory cAMP analogues (RpCPT-cAMP) confirmed that rolipram worked by stimulating PKA. These data suggest that PKA-activating agents, such as rolipram, can block secretion of TNF-alpha from macrophages by inhibiting T cell activation and expression of surface molecules.
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PMID:Regulation of IL-15-stimulated TNF-alpha production by rolipram. 1045 29

The beneficial roles of dietary fish oil in lowering serum TAG levels in animals and humans have been attributed in part to the high content of two n-3 polyunsaturated very long-chain FA, EPA, and DHA. Recent studies show that EPA induces mitochondrial beta-oxidation in hepatocytes, which might contribute to the systemic lipid-lowering effect. Whether EPA affects FA storage or oxidation in adipocytes is not clear. To investigate this possibility, 3T3-L1 adipocytes incubated with EPA (100 microM) for 24 h were assayed for beta-oxidation, carnitine palmitoyl transferase 1 (CPT-1) activity, protein, and mRNA expression of CPT-1. For comparison, cells treated with oleic acid, octanoic acid, and clofibrate, a synthetic ligand for peroxisome proliferator-activated receptor alpha were also analyzed. Mitochondria were isolated by differential centrifugation, and the mitochondrial membrane acyl chain composition was measured by GLC. EPA increased the oxidation of endogenous FA but did not inhibit lipogenesis. Oleic acid and clofibrate did not affect FA oxidation or lipogenesis, whereas octanoic acid suppressed the oxidation of endogenous FA and inhibited lipogenesis. Increased beta-oxidation by EPA was associated with increased CPT-1 activity but without changes in its mRNA and protein expression. EPA treatment increased the percentage of this FA in the mitochondrial membrane lipids. We suggest that EPA increased the activity of CPT-1 and beta-oxidation in adipocytes by altering the structure or dynamics of the mitochondrial membranes.
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PMID:Eicosapentaenoic acid, but not oleic acid, stimulates beta-oxidation in adipocytes. 1629

Recent studies have shown that dietary oxidised fats influence the lipid metabolism in rats by activation of PPARalpha. In this study, we investigated whether a mildly oxidised fat causes activation of PPARalpha in pigs which are non-proliferators like man. Eighteen pigs were assigned to two groups and received either a diet containing 90 g/kg of a fresh fat or the same diet with 90 g/kg of an oxidised fat prepared by heating for 24 h at 180 degrees C in a deep fryer. Pigs fed the oxidised fat had a higher peroxisome count, a higher activity of catalase and a higher mRNA concentration of mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase in the liver and a higher concentration of 3-hydroxybutyrate in plasma than pigs fed the fresh fat (P< 0.05). Hepatic mRNA concentrations of acyl-CoA oxidase and carnitine palmitoyltransferase- 1 tended to be increased in pigs fed the oxidised fat compared to pigs fed the fresh fat (P< 0.10). Pigs fed the oxidised fat, moreover, had higher mRNA concentrations of sterol regulatory element-binding protein (SREBP)-1 and its target genes acetyl-CoA carboxylase and stearoyl-CoA desaturase in the liver and higher mRNA concentrations of SREBP-2 and its target genes 3-hydroxy-3-methylglutary-CoA reductase and LDL receptor in liver and small intestine. In conclusion, this study shows that even a mildly oxidised fat causes activation of PPARalpha in the liver of pigs. Up-regulation of SREBP and its target genes in liver and small intestine suggests that the oxidised fat could stimulate synthesis of cholesterol and TAG in these tissues.
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PMID:Feeding of a deep-fried fat causes PPARalpha activation in the liver of pigs as a non-proliferating species. 1738 80

As tools for mechanistic studies on lipid metabolism, with the long-term goal of developing a drug for the treatment of lipid disorders, thia FA with the sulfur atom inserted at positions 3-9 from the carboxyl group were fed to male Wistar rats for 1 wk to determine their impact on key parameters in lipid metabolism and hepatic levels of thia FA metabolites. Thia FA with the sulfur atom in even positions decreased hepatic and cardiac mitochondrial beta-oxidation and profoundly increased hepatic and cardiac TAG levels. The plasma TAG level was unchanged and the hepatic acyl-CoA oxidase activity increased. In contrast, thia FA with the sulfur atom in odd positions, especially 3-thia FA, tended to increase hepatic and cardiac FA oxidation and acyl-CoA oxidase and carnitine palmitoyltransferase-II activities, and decreased the plasma TAG levels. The effects seem to be related to differences in the catabolic rate of the thia FA. Differences between the two groups of acids were also observed with respect to the regulation of genes involved in FA transport and catabolism. Feeding experiments with 3- and 4-thia FA in combination indicated that the 4-thia FA partly attenuated the effects of the 3-thia FA on mitochondrial FA oxidation and the hepatic TAG level. In summary, the position of the sulfur atom in the alkyl chain, especially whether it is placed in the even or odd position, is crucial for the biological effect of the thia FA.
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PMID:Thia fatty acids with the sulfur atom in even or odd positions have opposite effects on fatty acid catabolism. 1770 83

Flaxseed lignan secoisolariciresinol diglucoside (SDG) has been reported to prevent and alleviate lifestyle-related diseases including diabetes and hypercholesterolaemic atherosclerosis. This study assesses the effect of SDG on the development of diet-induced obesity in mice and the effect of the SDG metabolite enterodiol (END) on adipogenesis in 3T3-L1 adipocytes. We compared body weight, visceral fat weight, liver fat content, serum parameters, mRNA levels of lipid metabolism-related enzymes and adiponectin in mice fed either a low-fat diet (5 % TAG), high-fat diet (30 % TAG) or high-fat diet containing 0.5 and 1.0 % (w/w) SDG for 4 weeks. Administration of SDG to mice significantly reduced high-fat diet-induced visceral and liver fat accumulation, hyperlipaemia, hypercholesterolaemia, hyperinsulinaemia and hyperleptinaemia. SDG also suppressed sterol regulatory element binding protein 1c mRNA level in the liver and induced increases in the adiponectin mRNA level in the white adipose tissue and carnitine palmitoyltransferase I mRNA level in the skeletal muscle. Differentiated 3T3-L1 adipocytes were treated with 0, 5, 10 and 20 mumol/l END and then assayed for mRNA expression of adipogenesis-related genes and DNA binding activity of PPARgamma to the PPAR response element consensus sequence. END induced adipogenesis-related gene mRNA expression including adiponectin, leptin, glucose transporter 4 and PPARgamma, and induced PPARgamma DNA binding activity in 3T3-L1 adipocytes. In conclusion, SDG induced adiponectin mRNA expression and showed beneficial effects on lipid metabolism in diet-induced obesity in mice. Flaxseed lignans are suggested to regulate adipogenesis-related gene expressions through an increase in PPARgamma DNA binding activity.
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PMID:Flaxseed lignan attenuates high-fat diet-induced fat accumulation and induces adiponectin expression in mice. 1825 24


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