Differential metabolism and trafficking of sphingolipids in differentiated <i>versus</i> undifferentiated HT29 cells

Babià, T; Hulstaert, C.E.; Deweerd, H; Hoekstra, Dick

doi:10.1002/ijc.2910540519

Cited by 18 publications

(21 citation statements)

References 26 publications

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“…In accordance with these results, the cell-associated fluorescence after a 1 h incubation consisted almost entirely of C 6 -NBD-DH-Cer (98.5%; n ϭ 2) or C 6 -NBDCer (97.0%, n ϭ 2) in the case of the L,erythro isomers, while 19.9% (n ϭ 2) of C 6 -NBD-D,erythro-DH-Cer and 43.9% (n ϭ 2) of C 6 -NBD-D,erythro-Cer had been converted to (glyco)sphingolipid products. (17,23,30). Apparently, as shown in this study, (C 6 -NBD-)DH-Cer, which is the proposed precursor for (C 6 -NBD-)Cer biosynthesis (desaturation), can serve as a direct precursor for (C 6 -NBD-)-GlcCer and SM biosynthesis, without preceding desaturation.…”

Section: Fig 2-continuedsupporting

confidence: 57%

“…Ref. 23). For further analysis, individual C 6 -NBD-sphingolipids were scraped from the HPTLC plates and eluted from the silica by washing with 10 ml of CH 3 Cl/CH 3 OH (1:1, v/v), followed by 10 ml of CH 3 OH.…”

Section: Methodsmentioning

confidence: 99%

“…Both Saturated and Desaturated-C 6 -NBD-Cer, a short-chain fluorescent analog of ceramide, is a well established precursor for (glyco)sphingolipid biosynthesis (17,23,30). In HT29 Gϩ cells, a number of products are synthesized during a 24-h incubation with C 6 -NBD-Cer, the main products being C 6 -NBD-SM and C 6 -NBD-GlcCer, plus lesser amounts of C 6 -NBDGalCer, C 6 -NBD-LacCer and C 6 -NBD-CTH (see "Experimental Procedures"; cf.…”

Section: -Nbd-dh-cer Is Converted To Complex Sphingolipidsmentioning

confidence: 99%

“…Ref. 23). In this study, we examined the fate of a C 6 -NBD-analog of dihydroceramide (C 6 -NBD-DH-Cer, Fig.…”

Section: -Nbd-dh-cer Is Converted To Complex Sphingolipidsmentioning

confidence: 99%

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Dihydroceramide Biology

Kok¹,

Nikolova‐Karakashian²,

Klappe³

et al. 1997

Journal of Biological Chemistry

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This study utilized fluorescent analogs to characterize the intracellular transport and metabolism of dihydroceramide (DH-Cer), an intermediate in de novo sphingolipid biosynthesis. When 6-[N-(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]hexanoyl-DH-Cer (C 6 -NBD-DHCer) was incubated with HT29, NRK, BHK, or HL-60 cells, it was efficiently converted to dihydrosphingomyelin and dihydroglucosylceramide, and a number of other sphingolipids, with the nature of the products depending on the cell line. In addition, complex sphingolipids were formed that contained a desaturated (sphingosine) backbone, indicating that DH-Cer (and/or its metabolites) were substrates for the desaturase(s) that introduce the 4,5-trans double bond. Based on the kinetics and inhibitor studies, double bond addition did not appear to occur with the complex sphingolipids directly, but rather, during turnover and resynthesis. The conversion of C 6 -NBD-DH-Cer to more complex sphingolipids was highly stereoselective for the natural D,erythro isomer of C 6 -NBD-DH-Cer. Interestingly, the stereochemistry of the sphingoid base backbone also affected the localization of fluorescent sphingolipids: the D,erythro species appeared in the Golgi apparatus, whereas other stereo-isomers accumulated in the endoplasmic reticulum. In addition to C 6 -NBD-Cer and C 6 -NBD-DH-Cer, C 6 -NBD-4-D-hydroxy-DH-Cer gave rise to formation of complex sphingolipids and localized at the Golgi apparatus. These studies indicate that dihydroceramide is used as the initial backbone of complex (glyco)sphingolipids, perhaps to avoid build up of ceramide as an intermediate since this is such a potent bioactive compound. The stereoselectivity in transport and metabolism suggests that trafficking of ceramide is protein-directed rather than simply a consequence of vesicular membrane flow.

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Section: Fig 2-continuedsupporting

confidence: 57%

Section: Methodsmentioning

confidence: 99%

Section: -Nbd-dh-cer Is Converted To Complex Sphingolipidsmentioning

confidence: 99%

“…Ref. 23). In this study, we examined the fate of a C 6 -NBD-analog of dihydroceramide (C 6 -NBD-DH-Cer, Fig.…”

Section: -Nbd-dh-cer Is Converted To Complex Sphingolipidsmentioning

confidence: 99%

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Dihydroceramide Biology

Kok¹,

Nikolova‐Karakashian²,

Klappe³

et al. 1997

Journal of Biological Chemistry

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“…The Golgi complex localization of both NBD‐SLs after their internalization from the plasma membrane is independent of the state of neuronal differentiation. This finding is interesting because in HT29 human colon carcinoma cells, endocytosed NBD‐GlcCer accumulates in the Golgi complex only in undifferentiated cells (26,27). Although we do not know the physiological significance of internalizing lipids to the Golgi complex in neuronal cells, it is tempting to speculate that this could be a good source to supply the high demands of membrane transport in these highly dynamic cells.…”

Section: Discussionmentioning

confidence: 94%

Endocytosis of NBD‐Sphingolipids in Neurons: Exclusion from Degradative Compartments and Transport to the Golgi Complex

Babià

Ledesma

Saffrich

et al. 2001

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Sphingolipids are abundant constituents of neuronal membranes that have been implicated in intracellular signaling, neurite outgrowth and differentiation. Differential localization and trafficking of lipids to membrane domains contribute to the specialized functions. In non-neuronal cultured cell lines, plasma membrane short-chain sphingomyelin and glucosylceramide are recycled via endosomes or sorted to degradative compartments. However, depending on cell type and lipid membrane composition, short-chain glucosylceramide can also be diverted to the Golgi complex. Here, we show that NBD-labeled glucosylceramide and sphingomyelin are transported from the plasma membrane to the Golgi complex in cultured rat hippocampal neurons irrespective of the stage of neuronal differentiation. Golgi complex localization was confirmed by colocalization and Golgi disruption studies, and importantly did not result from conversion of NBD-glucosylceramide or NBD-sphingomyelin to NBD-ceramide. Double-labeling experiments with transferrin or wheat-germ agglutinin showed that NBD-sphingolipids are first internalized to early/recycling endosomes, and subsequently transported to the Golgi complex. The internalization of these two sphingolipid analogs was energy and temperature dependent, and their intracellular transport was insensitive to the NBD fluorescence quencher sodium dithionite. These results indicate that vesicles mediate the transport of internalized NBD-glucosylceramide and NBD-sphingomyelin to the Golgi complex.

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2010

Handbook of Biological Dyes and Stains

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Differential metabolism and trafficking of sphingolipids in differentiated versus undifferentiated HT29 cells

Cited by 18 publications

References 26 publications

Dihydroceramide Biology

Dihydroceramide Biology

Endocytosis of NBD‐Sphingolipids in Neurons: Exclusion from Degradative Compartments and Transport to the Golgi Complex

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