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

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

A Scaling Theory of Bifurcations in the Symmetric Weak-Noise Escape Problem

We consider the overdamped limit of two-dimensional double well systems perturbed by weak noise. In the weak noise limit the most probable fluctuational path leading from either point attractor to the separatrix (the most probable escape path, or MPEP) must terminate on the saddle between the two wells. However, as the parameters of a symmetric double well system are varied, a unique MPEP may bifurcate into two equally likely MPEP's. At the bifurcation point in parameter space, the activation kinetics of the system become non-Arrhenius. In this paper we quantify the non-Arrhenius behavior of a system at the bifurcation point, by using the Maslov-WKB method to construct an approximation to the quasistationary probability distribution of the system that is valid in a boundary layer near the separatrix. The approximation is a formal asymptotic solution of the Smoluchowski equation. Our analysis relies on the development of a new scaling theory, which yields `critical exponents' describing weak-noise behavior near the saddle, at the bifurcation point.Comment: LaTeX, 60 pages, 24 Postscript figures. Uses epsf macros to include the figures. A file in `uufiles' format containing the figures is separately available at ftp://platinum.math.arizona.edu/pub/papers-rsm/paperF/figures.uu and a Postscript version of the whole paper (figures included) is available at ftp://platinum.math.arizona.edu/pub/papers-rsm/paperF/paperF.p

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

Improved management of lysosomal glucosylceramide levels in a mouse model of type 1 Gaucher disease using enzyme and substrate reduction therapy

Gaucher disease is caused by a deficiency of the lysosomal enzyme glucocerebrosidase (acid βâ glucosidase), with consequent cellular accumulation of glucosylceramide (GLâ 1). The disease is managed by intravenous administrations of recombinant glucocerebrosidase (imiglucerase), although symptomatic patients with mild to moderate type 1 Gaucher disease for whom enzyme replacement therapy (ERT) is not an option may also be treated by substrate reduction therapy (SRT) with miglustat. To determine whether the sequential use of both ERT and SRT may provide additional benefits, we compared the relative pharmacodynamic efficacies of separate and sequential therapies in a murine model of Gaucher disease (D409V/null). As expected, ERT with recombinant glucocerebrosidase was effective in reducing the burden of GLâ 1 storage in the liver, spleen, and lung of 3â monthâ old Gaucher mice. SRT using a novel inhibitor of glucosylceramide synthase (Genzâ 112638) was also effective, albeit to a lesser degree than ERT. Animals administered recombinant glucocerebrosidase and then Genzâ 112638 showed the lowest levels of GLâ 1 in all the visceral organs and a reduced number of Gaucher cells in the liver. This was likely because the additional deployment of SRT following enzyme therapy slowed the rate of reaccumulation of GLâ 1 in the affected organs. Hence, in patients whose disease has been stabilized by intravenously administered recombinant glucocerebrosidase, orally administered SRT with Genzâ 112638 could potentially be used as a convenient maintenance therapy. In patients naïve to treatment, ERT followed by SRT could potentially accelerate clearance of the offending substrate.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/147062/1/jimd0281.pd

Studies of the action of ceramide-like substances ( d - and l -PDMP) on sphingolipid glycosyltransferases and purified lactosylceramide synthase

We have studied the effects of D -threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol ( D -PDMP) and its L -enantiomer on glycosphingolipids in cultured normal human kidney proximal tubular cells. We found that D -PDMP exerted a concentration-dependent reduction in the metabolic labelling and cellular levels of glucosylceramide (GlcCer), lactosylceramide (LacCer), and the globo-series glycosphingolipids, GbOse 3 Cer and GbOse 4 Cer. It also directly inhibited the activity of UDP-glucose:ceramide β1 → 4-glucosyltransferase (GlcT-1) and UDP-galactose: GlcCer β1 → 4 galactosyltransferase (GalT-2). In contrast, L -PDMP had opposite effects on the metabolic labelling of GlcCer, LacCer, and GbOse 3 Cer. The levels of GlcCer and LacCer were increased, while the labelling and level of GbOse 4 Cer were strongly reduced. Purified GalT-2 from human kidney was inhibited by D -PDMP and stimulated by L -PDMP. It appears likely that the different glycosphingolipid glycosyltransferases possess similar binding sites for the ceramide moiety, which are blocked by binding to D -PDMP and, in the case of GbOse 4 Cer synthase, by L -PDMP as well. The stimulatory effects of L -PDMP on GlcCer and LacCer synthases may be the result of binding to a modulatory site on the glycosyltransferases; in intact cells, the enzyme-analog complex may afford protection against the normal catabolic inactivation of the enzymes.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/45706/1/10719_2004_Article_BF00731481.pd

An Introduction to Sphingolipid Metabolism and Analysis by New Technologies

Sphingolipids (SP) are a complex class of molecules found in essentially all eukaryotes and some prokaryotes and viruses where they influence membrane structure, intracellular signaling, and interactions with the extracellular environment. Because of the combinatorial nature of their biosynthesis, there are thousands of SP subspecies varying in the lipid backbones and complex phospho- and glycoheadgroups. Therefore, comprehensive or “sphingolipidomic” analyses (structure-specific, quantitative analyses of all SP, or at least all members of a critical subset) are needed to know which and how much of these subspecies are present in a system as a step toward understanding their functions. Mass spectrometry and related novel techniques are able to quantify a small fraction, but nonetheless a substantial number, of SP and are beginning to provide information about their localization. This review summarizes the basic metabolism of SP and state-of-art mass spectrometric techniques that are producing insights into SP structure, metabolism, functions, and some of the dysfunctions of relevance to neuromedicine

The 3D OrbiSIMS—label-free metabolic imaging with subcellular lateral resolution and high mass-resolving power

We report the development of a 3D OrbiSIMS instrument for label-free biomedical imaging. It combines the high spatial resolution of secondary ion mass spectrometry (SIMS; under 200 nm for inorganic species and under 2 μm for biomolecules) with the high mass-resolving power of an Orbitrap (>240,000 at m/z 200). This allows exogenous and endogenous metabolites to be visualized in 3D with subcellular resolution. We imaged the distribution of neurotransmitters—gamma-aminobutyric acid, dopamine and serotonin—with high spectroscopic confidence in the mouse hippocampus. We also putatively annotated and mapped the subcellular localization of 29 sulfoglycosphingolipids and 45 glycerophospholipids, and we confirmed lipid identities with tandem mass spectrometry. We demonstrated single-cell metabolomic profiling using rat alveolar macrophage cells incubated with different concentrations of the drug amiodarone, and we observed that the upregulation of phospholipid species and cholesterol is correlated with the accumulation of amiodarone