Fluorescence assay of glucosylceramide glucosidase using NBD‐Cerebroside

A sensitive fluorometric assay for glucocerebroside β‐glucosidase [Dinur, T., Grabowski, G.A., Desnick, R.J., and Gatt, S. (1984)Anal. Biochem. 136, 223–234] has been reexamined. It was found that the lipids containing the NBD moiety (12‐[N‐methyl‐N‐(7‐nitrobenz‐2‐oxa‐1,3‐diazol‐4‐yl)] used for standardization of the assay are light‐sensitive and that the yield of fluorescent light is very sensitive to the composition of the solvent used in the fluorometric measurement. Some protection against fading could be obtained by adding a free‐radical trapping agent, Slow Fade. The fading of the free NBD‐acid, when used for standardization, could be prevented by adding ethanol to the solvent, but this reduced the fluorescence yield. It is recommended that some of the fluorescent substrate be enzymatically hydrolyzed completely to NBD‐ceramide, which can be utilized as the standard without the need to add ethanol. A warning about enzyme reaction rate stability with time is given, with a suggestion for ensuring constancy of activity.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/141148/1/lipd1052.pd

The phosphatidylinositol synthase of proximal tubule cells

Phosphatidylinositol (PI) is a precursor for an important class of phospholipids, the phosphatidylinositol polyphosphates. Because renal myo-inositol levels may vary under both physiological (e.g., antidiuretic) and pathophysiological (e.g., diabetic) conditions, the formation of PI from CDP-diacylglyceroI (CDP-DG) and myo-inositol via phosphatidylinositol synthase and the regulation of this enzyme have important implications for the cellular biology of renal epithelia. We sought to understand the role of PI synthase by determining its subcellular localization, kinetic properties and regulation in rabbit proximal tubule cells. Proximal tubule cells were isolated from New Zealand White rabbits. The subcellular synthesis of PI was assessed by [32P]orthophosphate labelling with subsequent subcellular fractionation. Labelling of PI was time-dependent and consistent with the rapid incorporation of 32PO4 into basolateral, brush-border, microsomal and nuclear fractions. Pulse-chase labelling of proximal tubule cells was consistent with the formation of PI in microsomal fraction of the proximal tubule cells in addition to both brush-border and basolateral membranes. Conversely, phosphatidylcholine, phosphatidylethanolamine and phosphatidylglycerol displayed radiolabelling patterns consistent with microsomal synthesis alone. The in situ formation of phosphatidylinositol was substantiated by the direct measurement of phosphatidylinositol synthase activity in basolateral, brush-border and microsomal fractions. The apparent Km values for myo-inositol were 0.32 +/- 0.19, 0.39 +/- 0.21 and 0.23 +/- 0.05 mM, and for CDP-DG were 0.12 +/- 0.02, 0.14 +/- 0.05 and 0.12 +/- 0.02 mM in basolateral, brush-border and microsomal fractions, respectively. Vmax values for phosphatidylinositol formation were slightly, but not significantly greater, in microsomal than for plasma membrane fractions. Moreover, based on enzymatic enrichment data, plasma membrane PI synthase activity could not be explained by microsomal cross-contamination alone. PI synthase activity was inhibited by co-incubation with PI without differences among the cellular fractions. Intracellular myo-inositol concentration in the proximal tubule cells as measured by gas-liquid chromatography was 20.5 mM, significantly greater than the apparent Km values for myo-inositol. In conclusion, the in situ synthesis of phosphatidylinositol occurs in several membrane fractions; the kinetic properties of phosphatidylinositol synthase appear to be similar in each fraction; and phosphatidylinositol synthase in proximal tubule cells is inhibited by its own formation product. These data suggest that myo-inositol concentration alone is unlikely to be an important regulator of the chemical mass of phosphatidylinositol at the levels of this polyol observed in rabbit kidney.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/28582/1/0000389.pd

Bradykinin-stimulated changes in inositol phosphate mass in renal papillary collecting tubule cells

The effect of bradykinin on changes in the chemical levels of -inositol polyphosphates in renal papillary collecting tubules was investigated. -inositol phosphate mass was determined by means of an enzymatic, fluorometric assay. Bradykinin induced increases in -inositol mono-, bis-, and trisphosphate which were both time and concentration dependent. Furthermore, the magnitude of the chemical levels of -inositol monophosphate formed were unlikely to be accounted for solely by the formation and degradation of -inositol trisphosphate. These observations are consistent with the concomitant hydrolysis of phosphatidylinositol and phosphatidylinositol bisphosphate. This study also confirms, in freshly isolated renal tubules, observations regarding bradykinin-induced phosphatidyl-inositol bisphosphate hydrolysis made previously in radiolabeled cultures.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/26674/1/0000218.pd

Role of diradylglycerol formation in H2O2 and lactoferrin release in adherent human polymorphonuclear leukocytes

Polymorphonuclear leukocytes (PMNs) adherent to fibrinogen exhibit a delay in the release of H2O2 in response to fMLP. Previously, we demonstrated that H2O2 release in adherent PMNs coincides with the exocytosis of lactoferrin‐containing specific granules and activation of phospholipase D (PLD). We also found that chelation of intracellular calcium blocked both lactoferrin and H2O2 release in stimulated PMNs in spite of the fact that adhesion and spreading remained normal. Since diradylglycerol (DRG) formation has been implicated in PMN secretion and oxidant release, we determined the effect of intracellular calcium chelation on PLD activation and DRG formation to ascertain whether DRG formation was coupled to lactoferrin and H2O2 release. We observed that chelation of intracellular calcium with bis‐(O‐aminophenoxy)‐ethanol‐N,N;N’‐ tetraacetic add (BAPTA) prevented PLD activation as monitored by inhibition of phosphatidylethanol formation. Formation of DRG derived from phosphatidic acid (PA) was also inhibited in the presence of BAPTA. Following the addition of the calcium ionophore ionomycin to the BAPTA‐treated PMNs, lactoferrin and H2O2 release was coincident with the onset of DRG formation. Also the addition of sn‐1,2‐didecanoylglycerol to the BAPTA‐treated PMNs stimulated them to release H2O2. Our studies support the hypothesis that DRG derived from PLD activation is required for degranulation of specific granules and associated H2O2 release from adherent PMNs. J. Leukoc. Biol. 56: 105–109; 1994.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/141596/1/jlb0105.pd

A turn in the road: How studies on the pharmacology of glucosylceramide synthase inhibitors led to the identification of a lysosomal phospholipase A 2 with ceramide transacylase activity

A series of inhibitors of glucosylceramide synthesis, the PDMP based family of compounds, has been developed as a tool for the study of sphingolipid biochemistry and biology. During the course of developing more active glucosylceramide synthase inhibitors, we identified a second site of inhibitory activity for PDMP and its structural homologues that accounted for the ability of the inhibitors to raise cell and tissue ceramide levels. This inhibitory activity was directed against a previously unknown pathway for ceramide metabolism, viz . the formation of 1- O -acylceramide. In this pathway the addition of a fatty acyl group to the primary hydroxyl of ceramide occurs through a transacylation with either phosphatidylethanolamine or phosphatidylcholine as a substrate. However, both in the absence and presence of ceramide, water serves as an acceptor for the fatty acid. Thus the enzyme may be considered to be a phospholipase A 2 . The enzyme is unique in that it has an acidic pH optimum and is localized to lysosomes by cell fractionation. More recently, the 1- O -acylceramide synthase has been purified, sequenced, and cloned. This phospholipase A 2 was discovered to be structurally homologous to lecithin cholesterol acyltransferase (LCAT). However, this phospholipase A 2 does not recognize cholesterol and lacks the defined lipoprotein-binding domain present in LCAT. We now refer to this enzyme as lysosomal phospholipase A 2 (LPLA 2 ). Although acidic phospholipase A 2 activities have been previously identified, LPLA 2 appears to be the first lysosomal PLA 2 to have been sequenced. This new phospholipase A 2 lacks an obvious and proven biological function. Published in 2004.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/45762/1/10719_2004_Article_5257241.pd

Effect of an Inhibitor of Glucosylceramide Synthesis on Cultured Human Keratinocytes

Glucosylceramide (GlcCer) is a major glycosphingolipid component of epidermis, which is thought to be related to the barrier function of skin permeability. However, the role of glycosphingolipids in keratinocyte growth and differentiation has not been fully clarified. It has been reported that D‐threo‐1‐phenyl‐2‐decanoylamino‐3‐morpholino‐1‐propanol (PDMP), an inhibitor of GlcCer synthase (EC 2.4.1.80), depletes cells of glycosphingolipids. This inhibitor has been used as a tool for elucidating their functions. In the present study, the effect of PDMP on cultured normal human keratinocytes was investigated. The cells were treated with various concentrations of PDMP. Forty‐eight hours later cell growth, thymidine incorporation, and lipid content were studied. The cell growth and the incorporation of thymidine into cells were inhibited by PDMP in a dose dependent manner. The synthesis of GlcCer was strongly inhibited by PDMP treatment, whereas no significant changes in ceramide level were observed. We concluded that GlcCer in epidermis may play an important role in regulating epidermal growth and suggested that PDMP may be beneficial for treating proliferative skin disorders in the future.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/111117/1/jde02353.pd

Sphingosine kinase 1–mediated inhibition of Fas death signaling in rheumatoid arthritis B lymphoblastoid cells

Objective It is becoming increasingly apparent that B cells play an important role in the pathogenesis of rheumatoid arthritis (RA). Due to the scarcity of B cells in RA, it has been technically difficult to functionally characterize B cell apoptosis in this disease. As a necessary first step to identify candidate aberrations, we investigated Fas-mediated signaling events in immortalized peripheral blood B lymphoblastoid cell lines (LCLs) from patients with RA and controls. Methods Cell death was determined by the MTS assay, and apoptosis was detected by the TUNEL assay and DNA laddering. Proteolytic activation of caspase 3 was determined by immunoblotting, and its enzymatic activity was determined by a fluorometric technique. Messenger RNA (mRNA) expression was quantified by real-time polymerase chain reaction (PCR) analysis. The functional role of sphingosine kinase (SPHK) was determined by measuring its enzymatic activity, by quantifying the levels of its product, sphingosine 1-phosphate (S1P), and by investigating the ability of the SPHK inhibitor N , N -dimethylsphingosine and isozyme-specific small interfering RNA (siRNA) oligonucleotides to reverse signaling aberrations. Results LCLs from patients with RA displayed disease-specific Fas-mediated signal transduction impairment with consequent resistance to cell death. RA LCLs displayed high constitutive SPHK activity and increased levels of S1P. Real-time PCR analysis showed higher SPHK-1 mRNA expression levels in RA patients compared with paired controls. Increased SPHK-1 (but not SPHK-2) mRNA levels were observed in synovial tissue from RA patients. Competitive inhibitors of SPHK reversed the resistance of RA LCLs to Fas-induced apoptosis. Additionally, resistance to Fas-mediated signaling was reversed by siRNA oligonucleotides specific for SPHK-1 but not by oligonucleotides specific for SPHK-2. Conclusion These findings demonstrate disease-specific resistance to Fas-mediated death signaling in patients with RA and implicate increased SPHK-1 activity as the cause of this aberration.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/49513/1/21635_ftp.pd

Studies on morpholinosphingolipids: Potent inhibitors of glucosylceramide synthase

Synthetic 1-morpholino-1-deoxyceramides were designed to inhibit glucosylceramide synthase. The most potent inhibitor 2a possesses the unnatural R,R-configuration of D-threo-sphingosine.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/31632/1/0000566.pd

Increased Hepatic Insulin Action in Diet-Induced Obese Mice Following Inhibition of Glucosylceramide Synthase

Obesity is characterized by the accumulation of fat in the liver and other tissues, leading to insulin resistance. We have previously shown that a specific inhibitor of glucosylceramide synthase, which inhibits the initial step in the synthesis of glycosphingolipids (GSLs), improved glucose metabolism and decreased hepatic steatosis in both ob/ob and diet-induced obese (DIO) mice. Here we have determined in the DIO mouse model the efficacy of a related small molecule compound, Genz-112638, which is currently being evaluated clinically for the treatment of Gaucher disease, a lysosomal storage disorder.DIO mice were treated with the Genz-112638 for 12 to 16 weeks by daily oral gavage. Genz-112638 lowered HbA1c levels and increased glucose tolerance. Whole body adiposity was not affected in normal mice, but decreased in drug-treated obese mice. Drug treatment also significantly lowered liver triglyceride levels and reduced the development of hepatic steatosis. We performed hyperinsulinemic-euglycemic clamps on the DIO mice treated with Genz-112638 and showed that insulin-mediated suppression of hepatic glucose production increased significantly compared to the placebo treated mice, indicating a marked improvement in hepatic insulin sensitivity.These results indicate that GSL inhibition in obese mice primarily results in an increase in insulin action in the liver, and suggests that GSLs may have an important role in hepatic insulin resistance in conditions of obesity

Substrate Reduction Augments the Efficacy of Enzyme Therapy in a Mouse Model of Fabry Disease

Fabry disease is an X-linked glycosphingolipid storage disorder caused by a deficiency in the activity of the lysosomal hydrolase α-galactosidase A (α-gal). This deficiency results in accumulation of the glycosphingolipid globotriaosylceramide (GL-3) in lysosomes. Endothelial cell storage of GL-3 frequently leads to kidney dysfunction, cardiac and cerebrovascular disease. The current treatment for Fabry disease is through infusions of recombinant α-gal (enzyme-replacement therapy; ERT). Although ERT can markedly reduce the lysosomal burden of GL-3 in endothelial cells, variability is seen in the clearance from several other cell types. This suggests that alternative and adjuvant therapies may be desirable. Use of glucosylceramide synthase inhibitors to abate the biosynthesis of glycosphingolipids (substrate reduction therapy, SRT) has been shown to be effective at reducing substrate levels in the related glycosphingolipidosis, Gaucher disease. Here, we show that such an inhibitor (eliglustat tartrate, Genz-112638) was effective at lowering GL-3 accumulation in a mouse model of Fabry disease. Relative efficacy of SRT and ERT at reducing GL-3 levels in Fabry mouse tissues differed with SRT being more effective in the kidney, and ERT more efficacious in the heart and liver. Combination therapy with ERT and SRT provided the most complete clearance of GL-3 from all the tissues. Furthermore, treatment normalized urine volume and uromodulin levels and significantly delayed the loss of a nociceptive response. The differential efficacies of SRT and ERT in the different tissues indicate that the combination approach is both additive and complementary suggesting the possibility of an improved therapeutic paradigm in the management of Fabry disease