D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol alters cellular cholesterol homeostasis by modulating the endosome lipid domains

D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP) is a frequently used inhibitor of glycosphingolipid biosynthesis. However, some interesting characteristics of D-PDMP cannot be explained by the inhibition of glycolipid synthesis alone. In the present study, we showed that d-PDMP inhibits the activation of lysosomal acid lipase by late endosome/lysosome specific lipid, bis(monoacylglycero)phosphate (also called as lysobisphosphatidic acid), through alteration of membrane structure of the lipid. When added to cultured fibroblasts, D-PDMP inhibits the degradation of low-density lipoprotein (LDL) and thus accumulates both cholesterol ester and free cholesterol in late endosomes/lysosomes. This accumulation results in the inhibition of LDL-derived cholesterol esterification and the decrease of cell surface cholesterol. We showed that D-PDMP alters cellular cholesterol homeostasis in a glycosphingolipid-independent manner using L-PDMP, a stereoisomer of D-PDMP, which does not inhibit glycosphingolipid synthesis, and mutant melanoma cell which is defective in glycolipid synthesis. Altering cholesterol homeostasis by D-PDMP explains the unique characteristics of sensitizing multidrug resistant cells by this drug.     

Glycosphingolipid accumulation inhibits cholesterol efflux via the ABCA1/apolipoprotein A-I pathway: 1-phenyl-2-decanoylamino-3-morpholino-1-propanol is a novel cholesterol efflux accelerator

Cellular glycosphingolipid (GSL) storage is known to promote cholesterol accumulation. Although physical interactions between GSLs and cholesterol are thought to cause intracellular cholesterol "trapping," it is not known whether cholesterol homeostatic mechanisms are also impaired under these conditions. ApoA-I-mediated cholesterol efflux via ABCA1 (ATP-binding cassette transporter A1) is a key regulator of cellular cholesterol balance. Here, we show that apoA-I-mediated cholesterol efflux was inhibited (by up to 53% over 8 h) when fibroblasts were treated with lactosylceramide or the glucocerebrosidase inhibitor conduritol B epoxide. Furthermore, apoA-I-mediated cholesterol efflux from fibroblasts derived from patients with genetic GSL storage diseases (Fabry disease, Sandhoff disease, and GM1 gangliosidosis) was impaired compared with control cells. Conversely, apoA-I-mediated cholesterol efflux from fibroblasts and cholesterol-loaded macrophage foam cells was dose-dependently stimulated (by up to 6-fold over 8 h) by the GSL synthesis inhibitor 1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP). Unexpectedly, a structurally unrelated GSL synthesis inhibitor, N-butyldeoxynojirimycin, was unable to stimulate apoA-I-mediated cholesterol efflux despite achieving similar GSL depletion. PDMP was found to up-regulate ABCA1 mRNA and protein expression, thereby identifying a contributing mechanism for the observed acceleration of cholesterol efflux to apoA-I. This study reveals a novel defect in cellular cholesterol homeostasis induced by GSL storage and identifies PDMP as a new agent for enhancing cholesterol efflux via the ABCA1/apoA-I pathway.     

D,L-Threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (DL-PDMP) increases endoplasmic reticulum stress, autophagy and apoptosis accompanying ceramide accumulation via ceramide synthase 5 protein expression in A549 cells

In A549 cells, the addition of D,L-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (DL-PDMP) led to marked autophagy with massive microtubule-associated protein 1 light chain 3B (LC3B)-II protein expression as an indication of autophagy and a steep decrease of p62 protein as a co-indication of autophagy. The addition of DL-PDMP caused massive autophagy with an increase of CAAT/enhancer binding protein homologous protein (CHOP) expression as the marker of endoplasmic reticulum (ER) stress, lactate dehydrogenase (LDH) release without caspase 3 activation and many autophagic vacuoles/devoid of a cell membrane on morphology. On the other hand, the addition of DL-PDMP caused an increase in cellular or subcellular ceramides (Cers), especially palmitoyl-Cer, based on de novo synthesis of Cer, and led to caspase-independent apoptosis. Marked increases of Cer levels in the nuclear envelope were observed 17 h after the addition. The elevations of Cer synthase activity and longevity-assurance homologue (LASS)5 protein expression were observed in subcellular fractions from 30 min until 2 h after the addition. However, the elevations of Cer synthase activity were independent of reactive oxygen species generation or cytochrome P450 4F2 activity. Since an increase in LASS5 protein expression in subcellular fraction occur in preference to the variation of LC3B-II protein expression via CHOP expression after the addition and Cer accumulation induced by the addition contributes to ER stress, it is thought that an elevation of Cer synthase activity via LASS5 protein expression associate to autophagy via CHOP expression (ER stress) with the addition.     

The inhibition of induction of microsomal monooxygenase activity by 1,3-diamino-2-propanol, an inhibitor of ornithine decarboxylase.

The p.o. administration of 1,3-diamino-2-propanol, an indirect inhibitor of ornithine decarboxylase, to rats and mice inhibited in a dose-dependent manner the induction of cytochrome P-450, benzo(a)pyrene hydroxylase, and 7-ethoxycoumarin O-deethylase by phenobarbital, β-naphtoflavone or Clophen C, a mixture of polychlorinated biphenyls. The results are consistent with the hypothesis that the induction of ornithine decarboxylase is a necessary step in the induction of microsomal monooxygenases.     

1-Phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) facilitates curcumin-induced melanoma cell apoptosis by enhancing ceramide accumulation, JNK activation, and inhibiting PI3K/AKT activation

The majority of metastatic melanomas are resistant to different chemotherapeutic agents, consequently, the search for novel anti-melanoma agents and adjuvant is urgent. Here, we found that 1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP), an inhibitor of glycosphingolipid biosynthesis, enhanced curcumin-induced cell growth inhibition and apoptosis in two melanoma cell lines (WM-115 and B16). PDMP facilitated curcumin-induced ceramide accumulation; the latter contributed to melanoma cell apoptosis. PDMP also dramatically enhanced curcumin-induced c-Jun N-terminal kinase activation, which was important to melanoma cell apoptosis. Meanwhile, curcumin plus PDMP treatment largely inhibited the activation of pro-survival PI3K/AKT signal pathway. In conclusion, PDMP-sensitized curcumin-induced melanoma cell growth inhibition and apoptosis in vitro due to changes of multiple signal events. Combining PDMP with curcumin may represent a new therapeutic intervention against melanoma.     

Liquid chromatography method for quantifying D-threo-1-phenyl-2-palmitoylamino-3-morpholino-1-propanol (D-threo-PPMP) in mouse plasma and liver

A high-performance liquid chromatography (HPLC) method was developed to measure levels of d-threo-1-phenyl-2-palmitoylamino-3-morpholino-1-propanol (d-threo-PPMP) in mouse plasma and liver. d-threo-PPMP was measured by HPLC with a Luna Pheny-Hexyl column (5 microm, 250 mm x 4.6 mm) employing UV detection at 210 nm using a mobile phase of potassium phosphate buffer (20mM, pH 3.0)-acetonitrile in a 45:55 (v/v) ratio. d-threo-1-phenyl-2-pentadecanoylamino-3-morpholino-1-propanol (PC15MP) was employed as an internal standard (IS). The lower limit of quantitation (LLOQ) was 0.3 microg/ml. The assay was linear over a concentration range of 0.3-10 microg/ml, with acceptable precision and accuracy. Assayed in plasma, the intra- and inter-day validation for all coefficients of variation (R.S.D.%) were found less than 15%. The method was applied to samples from athymic (nu/nu) mice treated with d-threo-PPMP by intraperitoneal injection. d-threo-PPMP levels of approximately 10-20 microg/ml ( approximately 20-40 microM) in plasma and approximately 45 microg/g in liver were obtained. The present method can be used to quantify d-threo-PPMP in mice for bioavailability and dose-response studies.     

Phosphoglucoisomerase-catalyzed interconversion of hexose phosphates: a model for the interconversion of D-[2-3H]glucose 6-phosphate and D-[1-3H]fructose 6-phosphate

Based on experimental data, a model is proposed for the interconversion of either unlabelled hexose phosphates or D-[2-3H]glucose 6-phosphate and D-[1-3H]fructose 6-phosphate in the reaction catalyzed by phosphoglucoisomerase. This model takes into account the known differences in maximal velocity and affinity for each substrate, the intramolecular transfer of tritium between C1 and C2, and the isotopic discrimination between unlabelled and tritiated esters. This model reveals that, in a close system characterized by the progressive detritiation of hexose phosphates, the concentration ratio of D-glucose 6-phosphate to D-fructose 6-phosphate is much higher with the tritiated than unlabelled esters, a paradoxical increase in the specific radioactivity of D-glucose 6-phosphate above its initial value being even observed during the initial period of exposure of D-[2-3H]glucose 6-phosphate to phosphoglucoisomerase. The extension of this model to an open system may be essential for the correct interpretation of radioactive data collected in intact cells exposed to D-[2-3H]glucose.     

Phenotypic changes in 3T3 cells associated with the change of sphingolipid synthesis by a ceramide analog, 2-decanoylamino-3-morpholino-1-phenylpropanol (compound RV538)

A culture of BALB/c 3T3 cells grown in the presence of 40 microM of the ceramide analog compound RV538 (2-decanolyamino-3-morpholino-1-phenylpropanol) for several passages caused a substantial decrease in the level of all glycosphingolipids and an accumulation of ceramide and sphingomyelin. Associated with these chemical changes of sphingolipid composition and metabolism, the following phenotypic changes were observed: (i) loss of the cobblestone appearance at cell density saturation and development of fibroblastic appearance with partial overlapping of cells; (ii) reduction of cell growth rate; (iii) enhanced production of lactic acid; (iv) enhanced rate of glucose transport; and (v) higher incidence of large colony formation with infiltrating appearance in soft agar. Cell morphology changes, lactate production, and enhanced sugar uptake were reversed by co-culturing cells with gangliosides, particularly trisialogangliosides. Thus, these phenotypic changes mimicking those of oncogenically transformed cells are closely related to the blocked synthesis of glycolipids in these cells, whereas other changes may be caused by an accumulation of ceramide and sphingomyelin.     

Labelling of lipids by D-[1-14C]glucose, D-[6-14C]glucose and D-[3-3H]glucose in pancreatic islets from normal and GK rats

In pancreatic islets prepared from either normal or GK rats and incubated at either low (2.8 mM) or high (16.7 mM) D-glucose concentration, the labelling of both lipids and their glycerol moiety is higher in the presence of D-[1-¹⁴C]glucose than D-[6-¹⁴C]glucose. The rise in D-glucose concentration augments the labelling of lipids, the paired ¹⁴C/³H ratio found in islets exposed to both D-[1-¹⁴C]glucose or D-[6-¹⁴C]glucose and D-[3-³H]glucose being even slightly higher at 16.7 mM D-glucose than that found, under otherwise identical conditions, at 2.8 mM D-glucose. Such a paired ratio exceeds unity in islets exposed to D-[1-¹⁴C]glucose. The labelling of islet lipids by D-[6-¹⁴C]glucose is about 30 times lower than the generation of acidic metabolites from the same tracer. These findings indicate (i) that the labelling of islet lipids accounts for only a minor fraction of D-glucose catabolism in pancreatic islets, (ii) a greater escape to L-glycerol-3-phosphate of glycerone-3-phosphate generated from the C₁-C₂-C₃ moiety of D-glucose than D-glyceraldehyde-3-phosphate produced from the C₄-C₅-C₆ moiety of the hexose, (iii) that only a limited amount of [3-³H]glycerone 3-phosphate generated from D-[3-³H]glucose is detritiated at the triose phosphate isomerase level before being converted to L-glycerol-3-phosphate, and (iv) that a rise in D-glucose concentration results in an increased labelling of islet lipids, this phenomenon being somewhat more pronounced in the case of D-[1-¹⁴C]glucose or D-[6-¹⁴C]glucose rather than D-[3-³H]glucose.     

Metabolism of 2-deoxy-2-fluoro-D-[3H]glucose and 2-deoxy-2-fluoro-D-[3H]mannose in yeast and chick-embryo cells.

2-Deoxy-2-fluoro-D-[3H]glucose and 2-deoxy-2-fluoro-D-[3H]mannose have been prepared by tritiation of the corresponding unlabeled 2-fluoro sugars. The tritiated 2-fluoro sugars are phosphorylated and activated by UTP and by GTP to yield UDP-2-deoxy-2-fluoro-D-[3H]glucose, UDP-2-deoxy-2-fluoro-D-[3H]mannose, GDP-2-deoxy-2-fluoro-D-[3H]glucose and GDP-2-deoxy-2-fluoro-D-[3H]mannose in both cell types. The nucleotide derivatives could also be labeled in the nucleotide moiety by feeding the cells with [14C]uridine or [14C]guanosine in the presence of unlabeled 2-fluoro sugar. No evidence was obtained for metabolic steps in which the six-carbon chain of 2-fluoro sugars was not preserved. No epimerisation of the label to 2-deoxy-2-fluoro-D-[3H]galactose could be observed by radioactive gas-liquid chromatography of the enzymatic cleavage products of the different 2-fluoro sugar metabolites isolated from either cell type. Yeast and chick embryo cells both incorporate 2-deoxy-2-fluoro-D-[3H]glucose and 2-deoxy-2-fluoro-D-[3H]mannose specifically into glycoproteins, although this incorporation is very low when compared to the incorporation of 2-deoxy-D-[3H]glucose.     

Mathematical modelling for the generation of L-[3-2H,3-13C]lactic acid isotopomers by erythrocytes exposed to either D-[1-13C]glucose or D-[6-13C]glucose in the presence of2H2O

Cytosolic purine nucleoside phosphorylase (PNPase) is a well known, and described enzyme which exists in a variety of organisms, both procaryotic and eucaryotic. More recently this enzyme was found in bovine liver mitochondria. The mitochondrial purine nucleoside phosphorylase was purified 63 fold and has a molecular weight of 48–60 kD. From Lineweaver-Burk plots apparent K_m's of 23M for inosine, 42 M for deoxyinosine, 40 M for phosphate, 2 M for hypoxanthine, and 163 M for ribose-1-phosphate were calculated. Both 8-aminoguanosine (K_i=0.5 M) and araG (K_i=381 M) are inhibitors of the enzyme. The protein's isoelectric point (pI) was calculated at a pH of 4.2. Preliminary immunological work showed no cross-reactivity between epitopes on the mitochondrial protein and those on PNPase from human erythrocytes. The apparent K_m's calculated for the mitochondrial enzyme are,with the exception of that using hypoxanthine, within the range commonly associated with K_m's from the cytosolic species. The mitochondrial enzyme's molecular weight and pI are less than normally described. The enzyme's isolation from mitochondria, together with several unique characteristics, suggest that it is a separate protein from that found in the cytosol.     

Conversion of erythro-D-sphinganine to its [1-2H1] and [1-3H1] derivatives

A convenient chemical synthesis of erythro-D-[1-2H1] sphinganine and erythro-D-[1-3H1]sphinganine is described. The approach utilizes a stereospecific starting material (natural sphinganine prepared from bovine brain sphingomyelin) and applies a sequence of selective protection of functional groups yielding 2-acetamido-3-O-benzoyloctadecan-1-ol. Oxidation of the primary alcohol to an aldehyde followed by NaB2H4 or NaB3H4 reduction and hydrolysis of the protective groups yields erythro-D-[1-2H1]sphinganine or erythro-D-[1-3H1]sphinganine. The synthetic intermediates and isotopically labeled sphinganines are characterized by infrared analysis, 1H-nuclear magnetic resonance, optical rotation, and gas-liquid radiochromatographic and mass spectral fragmentation analyses. The [1-2H1] and [1-3H1] derivatives were obtained with overall yields (and isotope enrichments) of 11% (min. 84 mol% 2H1) and 8% (60 mCi/mmol), respectively.     

Underestimation of D-glucose phosphorylation as measured by 3H2O production from D-[2-3H]glucose

In rat pancreatic islets, tumoral islet cells (RINm5F line), parotid gland, and in human erythrocytes, but not in rat hepatocytes, the production of 3H2O from D-[2-3H]glucose is 20-30% lower than from D-[5-3H]glucose. This coincides with the production of tritiated lactic acid from D-[2-3H]glucose and may be attributable to an intramolecular hydrogen transfer in the phosphoglucoisomerase reaction. It is concluded that the production of 3H2O from D-[2-3H]glucose is not a reliable tool to assess the total rate of hexose phosphorylation.     

Chemoenzymatic synthesis of stable isotope labeled UDP-N-[2H]-acetyl-glucosamine and [2H]-acetyl-chitooligosaccharides

Labeled UDP-GlcNAc and chitooligosaccharides should be powerful tools for studies of N-acetylglucosaminyltransferase such as chitin synthases. We describe here a rapid, inexpensive and a common strategie for the chemoenzymatic synthesis of uridine 5′-diphospho-N-[²H]-acetyl-glucosamine and the chemical preparation of N-[²H]-acetyl chitooligosaccharides (from 2 to 5 mers). Deuterated UDP-GlcNAc analogue was tested as chitin synthase substrate and it exhibited an incorporation level in chitin as the natural substrate. Deuterium labeling of carbohydrates present different advantages: it is a stable isotope and allows glycosyltransferase mechanism studies by a mass spectrometry approach.     

Improved and efficient synthesis of 1alpha-hydroxy-[6-(2)H] and 1alpha-hydroxy-[6,19,19-(2)H]vitamin D3 derivatives.

Improved and efficient procedures for deuterium-labeling at the 6,19,19 positions of 1alpha-hydroxyvitamin D3 derivatives via its sulfur dioxide-adduct by using a base-catalyzed H-D exchange reaction are described. Application of the known procedure using tBuOK/DMF-D2O, which is effective for labeling vitamin D3 derivatives, to 1alpha-hydroxy compounds gave only poor results because of isomerization and decomposition. We found that this procedure is improved by the use of iPrONa/iPrOD. During this study, we also found that the 6-monodeuterated product was selectively obtained when MeONa/CD3OD was employed instead of iPrONa/iPrOD. On the other hand, simple addition of 1,3-dimethyl-2-imidazolidinone as a co-solvent to the above conditions was effective for 1alpha,25-dihydroxy compounds. These improved procedures were successfully applied to the synthesis of 1alpha-hydroxy-[6,19,19-(2)H]vitamin D3 derivatives 4 and 1alpha-hydroxy-[6-(2)H]vitamin D3 derivatives 6 from the corresponding 1alpha-hydroxyvitamin D3 derivatives 1 via its sulfur dioxide-adducts 2, 3 and 5 in good over-all yield with high deuterium incorporation.     

Synthesis of 25-hydroxy-[6,19,19'-2H3]vitamin D3 and 1 alpha,25-dihydroxy-[6,19,19'-2H3]vitamin D3.

Synthesis of polydeuterated analogs of 25-hydroxyvitamin D3 and 1 alpha,25-dihydroxy vitamin D3 are described. These analogs, containing stable isotope atoms at metabolically stable positions, are potentially useful in studies involving catabolism of hydroxylated metabolites of vitamin D3.     

Regulation of agonist and antagonist binding to striatal D-1 dopamine receptors: studies using the selective D-1 antagonist [3H]SK&F R-83566

The potent and D-1 versus D-2 selective dopamine receptor antagonist, SK&F R-83566, was radiolabelled with tritium and was used as a radioligand for examination of D-1 receptors in rat striatum. Binding of the radioligand was stereoselective, saturable and reversible. In homogenates of rat striatum, nonspecific binding of the radioligand was less than 5% of total binding, the KD was 1.1 +/- 0.2 nM and the Bmax was 1130 +/- 70 fmoles/mg protein. Results of competition binding analyses yielded a pharmacological profile that was characteristic of dopamine D-1 receptor interaction. Competition studies of dopamine agonists against the potent antagonist radioligand indicated multiple affinities of agonist binding to the D-1 receptor. Displacement was best fit to a two-site model of ligand binding and high and low affinities were subject to regulation by guanine, but not adenine, nucleotides. Antagonist binding was not complex and was unaffected by guanine nucleotides. The role of monovalent cations in regulating D-1 receptor binding was evaluated by comparing effects of Na+, Li+, and K+ on binding of the antagonist [3H]SK&F R-83566 and the agonist [3H]fenoldopam (SK&F 82526). Whereas agonist binding was reduced in a concentration dependent fashion by monovalent cations with a ranking of potency Li+ greater than Na+ greater than K+, antagonist binding was enhanced by the cation Na+ but little affected by Li+ or K+. This effect of relatively low concentrations of Na+ to decrease agonist binding and increase antagonist binding suggests similarities between the D-1 receptor which is positively-coupled to adenylate cyclase and other receptors, e.g. alpha 2 adrenergic receptors, which are negatively-coupled to adenylate cyclase.