C 2 -and C 6 -ceramides (N-acetylsphingosine and N-hexanoylsphingosine, respectively) abolished the stimulation of DNA synthesis by sphingosine 1-phosphate in rat fibroblasts. This inhibition by ceramide was partially prevented by insulin. C 2 -ceramide did not alter the stimulation of DNA synthesis by insulin and decreased the sphingosine-induced stimulation by only 16%. The ceramides did not significantly modify the actions of sphingosine or sphingosine 1-phosphate in decreasing cAMP concentrations. C 2 -and C 6 -ceramides blocked the activation of phospholipase D by sphingosine 1-phosphate, and this inhibition was not affected by insulin. Okadaic acid decreased the activation of phospholipase D by sphingosine 1-phosphate and did not reverse the inhibitory effect of C 2 -ceramide on this activation. Therefore, this effect of C 2 -ceramide is unlikely to involve the stimulation of phosphoprotein phosphatase activity. Sphingosine did not activate phospholipase D activity significantly after 10 min. C 2 -ceramide stimulated the conversion of exogenous [ 3 H]sphingosine 1-phosphate to sphingosine and ceramide in fibroblasts. Ceramides can inhibit some effects of sphingosine 1-phosphate by stimulating its degradation via a phosphohydrolase that also hydrolyzes phosphatidate. Furthermore, C 2 -and C 6 -ceramides stimulated ceramide production from endogenous lipids, and this could propagate the intracellular signal. This work demonstrates that controlling the production of ceramide versus sphingosine and sphingosine 1-phosphate after sphingomyelinase activation could have profound effects on signal transduction.

Incubation of 3T3-L1 adipocytes with C2- and C6-ceramides (N-acetyl- and N-hexanoylsphingosines) but not dihydro-C2-ceramide increased 2-deoxyglucose uptake in the absence of insulin. This effect was inhibited by PD 98059, LY 294002, and rapamycin, which block the activation of mitogen-activated protein kinase, phosphatidylinositol (PI) 3-kinase, and ribosomal S6 kinase, respectively. Long-term increases in PI 3-kinase activity associated with insulin receptor substrate 1 (IRS-1) increased GLUT1 and GLUT4 concentrations in plasma membranes. This together with increased GLUT1 (but not GLUT4) synthesis explains the increase in non-insulin-dependent glucose uptake. C2-ceramide inhibited insulin-stimulated glucose uptake after 2 h by decreasing insulin-induced translocation of GLUT1 and GLUT4 to plasma membranes. This occurred when there was no increase in basal glucose uptake or decrease in activation of IRS-1 or PI 3-kinase. Incubation for 24 h with tumor necrosis factor-alpha (TNF-alpha) but not C2-ceramide decreased the concentration and insulin-induced tyrosine phosphorylation of IRS-1 in this experimental system. Cell-permeable ceramides mimic some effects of TNF-alpha, especially in stimulating basal glucose uptake. We identified a site for inhibiting insulin-stimulated glucose uptake that is downstream of PI 3-kinase. Our work provides further mechanisms for the effects of TNF-alpha and ceramides in increasing non-insulin-dependent glucose uptake and decreasing insulin-stimulated uptake in vivo.

The mechanism of inhibition of phospholipase D (PLD) by ceramides was determined using granulocytes differentiated from human promyelocytic leukemic (HL-60) cells. In a cell-free system, hydrolysis of phosphatidylcholine by membrane-bound PLD depended upon phosphatidylinositol 4,5-bisphosphate, guanosine 5-3-O-(thio)triphosphate) (GTP␥S), and cytosolic factors including ADP-ribosylating factor (ARF) and RhoA. C 2 -(N-acetyl-), C 8 -(N-octanoyl-), and long-chain ceramides, but not dihydro-C 2 -ceramide, inhibited PLD activity. Apyrase or okadaic acid did not modify the inhibition of PLD by ceramides, indicating that the effect in the cellfree system was unlikely to be dependent upon a ceramide-stimulated kinase or phosphoprotein phosphatases. C 2 -and C 8 -ceramides prevented the GTP␥S-induced translocation of ARF1 and RhoA from the cytosol to the membrane fraction. In whole cells, C 2 -ceramide, but not dihydro-C 2 -ceramide, inhibited the stimulation of PLD by N-formylmethionylleucylphenylalanine and decreased the amounts of ARF1, RhoA, CDC42, Rab4, and protein kinase C-␣ and -␤ 1 that were associated with the membrane fraction, but did not alter the distribution of protein kinase C-⑀ and -. It is concluded that one mechanism by which ceramides prevent the activation of PLD is inhibition of the translocation to membranes of G-proteins and protein kinase C isoforms that are required for PLD activity. PLD1 in mammalian cells plays a key role in signal transduction and its activation occurs in a wide range of cell types in response to hormones and growth factors. Several components such as G-proteins, PKC, and Ca 2ϩ are involved in regulating PLD (for review, see Refs. 1 and 2). PLD catalyzes the hydrolysis of cell phospholipids, mainly PC, resulting in the formation of PA, which may also be the precursor of lysoPA. These two lipids are bioactive, and they have many effects that are similar. PA or lysoPA been reported to stimulate the respiratory burst in neutrophils (3), monoacylglycerol acyltransferase (4), phospholipase C␥ (5), phosphatidylinositol-4-phosphate kinase (6), PKC- (7), and the Ras-Raf-mitogen-activated protein kinase pathway (8) and to inhibit adenylate cyclase (9). PA may also be dephosphorylated by phosphatidate phosphohydrolase to diacylglycerol (10), a well characterized activator of PKC (2).In cell-free assays, the activation of membrane-associated PLD by GTP␥S is dependent on the presence of both membranes and cytosol components. The latter consist of small G-proteins of the Ras superfamily, such as ARF (11, 12), RhoA (13, 14), and CDC42 (15, 16). ARF was first identified as a cofactor necessary for the ADP-ribosylation of the ␣-subunit of heterotrimeric G-proteins, i.e. G s , by cholera toxin (17). ARF has also been implicated in vesicular transport in the Golgi (18) and in endocytosis (19). ARF-stimulated PLD has been partially purified from HL-60 cell membranes, and this stimulation was dependent on the presence of PIP 2 (12). Subsequently, ARF-stimulated PLD was separated from oleate-stim...

Concentrations of the bioactive lipids, phosphatidate and diacylglycerol, increased with time in culture in ras-and tyrosine kinase (fps)-transformed ®broblasts but not in control ®broblasts. On Day 3, diacylglycerol and phosphatidate concentrations were about 3.3-and 5.5-fold higher respectively in the ras-transformed compared to control ®broblasts. These concentrations in fpstransformed ®broblasts were increased about twofold. The changes in phosphatidate and diacylglycerol resulted from enhanced phospholipid turnover rather than from synthesis de novo. The increased ratio of phosphatidate to diacylglycerol is explained by decreased activities of two distinct phosphatidate phosphohydrolases and increased diacylglycerol kinase in ras-transformed ®bro-blasts. Ceramide concentrations were about 2.5-and threefold higher in the fps-and ras-transformed cells respectively on Day 3 compared to the controls. Incubating control ®broblasts from Days 1 to 3 with phosphatidylcholine-speci®c phospholipase C increased diacylglycerol, phosphatidate and ceramide concentrations, and decreased Mg 2+ -independent-phosphatidate phosphohydrolase activity. 8-(4-chlorophenylthio)-cAMP had a cytostatic e ect in ras-transformed cells, it decreased the concentrations of phosphatidate and diacylglycerol, but increased that of ceramide. The consequences of increased ceramide and phosphatidate concentrations in ras-transformed cells are discussed in relation to signal transduction, cell division and the transformed phenotype.

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