Adipocytes package incoming fatty acids into triglycerides and other glycerolipids, with only a fraction spilling into a parallel biosynthetic pathway that produces sphingolipids. Herein, we demonstrate that subcutaneous adipose tissue of type 2 diabetics contains considerably more sphingolipids than non-diabetic, BMI-matched counterparts. Whole-body and adipose tissue-specific inhibition/deletion of serine palmitoyltransferase (Sptlc), the first enzyme in the sphingolipid biosynthesis cascade, in mice markedly altered adipose morphology and metabolism, particularly in subcutaneous adipose tissue. The reduction in adipose sphingolipids increased brown and beige/brite adipocyte numbers, mitochondrial activity, and insulin sensitivity. The manipulation also increased numbers of anti-inflammatory M2 macrophages in the adipose bed and induced secretion of insulin-sensitizing adipokines. By comparison, deletion of serine palmitoyltransferase from macrophages had no discernible effects on metabolic homeostasis or adipose function. These data indicate that newly synthesized adipocyte sphingolipids are nutrient signals that drive changes in the adipose phenotype to influence whole-body energy expenditure and nutrient metabolism.

Previous work has led to the identification of inhibitors of glucosylceramide synthase, the enzyme catalyzing the first glycosylation step in the synthesis of glucosylceramide-based glycosphingolipids. These inhibitors have two identified sites of action: the inhibition of glucosylceramide synthase, resulting in the depletion of cellular glycosphingolipids, and the inhibition of 1-O-acylceramide synthase, resulting in the elevation of cell ceramide levels. A new series of glucosylceramide synthase inhibitors based on substitutions in the phenyl ring of a parent compound, 1-phenyl-2-palmitoylamino-3-pyrrolidino-1-propanol (P4), was made. For substitutions of single functional groups, the potency of these inhibitors in blocking glucosylceramide synthase was primarily dependent upon the hydrophobic and electronic properties of the substituents. An exponential relationship was found between the IC 50 of each inhibitor and the sum of derived hydrophobic () and electronic () parameters. This relationship demonstrated that substitutions that increased the electron-donating characteristics and decreased the lipophilic characteristics of the homologues enhanced the potency of these compounds in blocking glucosylceramide formation. A novel compound was subsequently designed and observed to be even more active in blocking glucosylceramide formation. This compound, D-threo-4-hydroxy-P4, inhibited glucosylceramide synthase at an IC 50 of 90 nM. In addition, a series of dioxane substitutions was designed and tested. These included 3,4-methylenedioxyphenyl-, 3,4-ethylenedioxyphenyl-, and 34-trimethylenedioxyphenyl-substituted homologues. D-threo-3,4-Ethylenedioxy-P4-inhibited glucosylceramide synthase was comparably active to the p-hydroxy homologue. 4-Hydroxy-P4 and ethylenedioxy-P4 blocked glucosylceramide synthase activity at concentrations that had little effect on 1-O-acylceramide synthase activity. These novel inhibitors resulted in the inhibition of glycosphingolipid synthesis in cultured cells at concentrations that did not significantly raise intracellular ceramide levels or inhibit cell growth. GlcCer1 is the precursor of hundreds of different glycosphingolipids. This cerebroside is synthesized from uridine diphosphate-glucose and ceramide by a glucosyltransferase, GlcCer synthase. GlcCer-based sphingolipids have been identified as important mediators of a variety of cellular functions, including proliferation, differentiation, development, and cell-cell recognition (1). The (R,R)-(D-threo)-isomer of 1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) and its homologues are potent inhibitors of GlcCer synthase. These compounds have been used extensively to study the metabolism and function of glycosphingolipids in living cells (2-6).In previously reported work, a series of PDMP homologues and analogues was synthesized (4). Replacing the decanoyl moiety with a palmitoyl moiety enhanced the effectiveness of PDMP. In addition, replacing the morpholino ring with a pyrrolidino ring, forming DL-threo-1-phenyl-2-palmito...

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Fabry disease, an X-linked systemic vasculopathy, is caused by a deficiency of α-galactosidase A resulting in globotriaosylceramide (Gb 3 ) storage in cells. The pathogenic role of Gb 3 in the disease is not known. Based on previous work, we tested the hypothesis that accumulation of Gb 3 in the vascular endothelium of Fabry disease is associated with increased production of reactive oxygen species (ROS) and increased expression of cell adhesion molecules. Gb 3 loading resulted in increased intracellular ROS production in cultured vascular endothelial cells in a dose-dependent manner. Increased Gb 3 also induced expression of intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and E-selectin. Reduction of endogenous Gb 3 by treatment of the cells with an inhibitor of glycosphingolipid synthase or α-galactosidase A led to decreased expression of adhesion molecules. Plasma from Fabry patients significantly increased ROS generation in endothelial cells when compared with plasma from non-Fabry controls. This effect was not influenced by reduction of intracellular Gb 3 . This study provided direct evidence that excess intracellular Gb 3 induces oxidative stress and up-regulates the expression of cellular adhesion molecules in vascular endothelial cells. In addition, other factors in patient's plasma may also contribute to oxidative stress in Fabry vascular endothelial cells.

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