Benzoxazolone Carboxamides: Potent and Systemically Active Inhibitors of Intracellular Acid Ceramidase

Pizzirani, Daniela; Bach, Anders; Realini, Natalia; Armirotti, Andrea; Mengatto, Luisa; Bauer, Inga; Girotto, Stefania; Pagliuca, Chiara; Vivo, Marco De; Summa, Maria; Ribeiro, Alison; Piomelli, Daniele

doi:10.1002/ange.201409042

Cited by 16 publications

(33 citation statements)

References 29 publications

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“…Sphingosine, the central substrate of SphK2 in hepatocytes, is mainly produced via ASAH1-mediated hydrolysis of ceramides (41). By blocking this pathway, the ceramidase inhibitor ARN14974 is known to elevate the ceramide level and decrease the sphingosine level in the cell (28). Interestingly, both ARN14974 and siRNAmediated knockdown of ASAH1 significantly rescued insulin sensitivity in SphK2-deficient hepatocytes ( Fig.…”

Section: Discussionmentioning

confidence: 90%

“…To segregate the impacts of ceramides and sphingosine, we treated cells with fumonisin b1 and ARN14974. Fumonisin b1 is a specific inhibitor of ceramide synthase, which reduces ceramides but increases sphingosine levels in cells (27), while ARN14974 is a novel inhibitor of acid ceramidase (ASAH1), which explicitly blocks ceramide conversion to sphingosine and thus reduces sphingosine content (28). Fumonisin b1 up to 50 μM failed to reverse Akt activation in SphK2 knockdown cells (Fig.…”

Section: Sphingosine Is Primarily Responsible For the Effect Of Sphk2 Onmentioning

confidence: 88%

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Regulation of hepatic insulin signaling and glucose homeostasis by sphingosine kinase 2

Aji

Huang

et al. 2020

Proc. Natl. Acad. Sci. U.S.A.

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Sphingolipid dysregulation is often associated with insulin resistance, while the enzymes controlling sphingolipid metabolism are emerging as therapeutic targets for improving insulin sensitivity. We report herein that sphingosine kinase 2 (SphK2), a key enzyme in sphingolipid catabolism, plays a critical role in the regulation of hepatic insulin signaling and glucose homeostasis both in vitro and in vivo. Hepatocyte-specific Sphk2 knockout mice exhibit pronounced insulin resistance and glucose intolerance. Likewise, SphK2-deficient hepatocytes are resistant to insulin-induced activation of the phosphoinositide 3-kinase (PI3K)-Akt-FoxO1 pathway and elevated hepatic glucose production. Mechanistically, SphK2 deficiency leads to the accumulation of sphingosine that, in turn, suppresses hepatic insulin signaling by inhibiting PI3K activation in hepatocytes. Either reexpressing functional SphK2 or pharmacologically inhibiting sphingosine production restores insulin sensitivity in SphK2-deficient hepatocytes. In conclusion, the current study provides both experimental findings and mechanistic data showing that SphK2 and sphingosine in the liver are critical regulators of insulin sensitivity and glucose homeostasis.

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Section: Discussionmentioning

confidence: 90%

Section: Sphingosine Is Primarily Responsible For the Effect Of Sphk2 Onmentioning

confidence: 88%

Regulation of hepatic insulin signaling and glucose homeostasis by sphingosine kinase 2

Aji

Huang

et al. 2020

Proc. Natl. Acad. Sci. U.S.A.

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“…Heck293-WT cells, Hek293 cells overexpressing hAC1 (Hek293-hAC1) [19], and Hek293 cells overexpressing human NAAA (Hek293-hNAAA) [20] were plated in 150-mm dishes (5 Â 10 6 cells/ dish) and cultured in complete DMEM containing 10% fetal bovine serum, 2 mM L-glutamine, and antibiotics (100 U/ml penicillin and streptomycin) at 37 C and 5% CO 2 . Cell media were collected 48 h after incubation.…”

Section: Cell Culturesmentioning

confidence: 99%

A simple and accurate protocol for absolute polar metabolite quantification in cell cultures using quantitative nuclear magnetic resonance

Goldoni

Beringhelli

Rocchia

et al. 2016

Analytical Biochemistry

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a b s t r a c tAbsolute analyte quantification by nuclear magnetic resonance (NMR) spectroscopy is rarely pursued in metabolomics, even though this would allow researchers to compare results obtained using different techniques. Here we report on a new protocol that permits, after pH-controlled serum protein removal, the sensitive quantification (limit of detection [LOD] ¼ 5À25 mM) of hydrophilic nutrients and metabolites in the extracellular medium of cells in cultures. The method does not require the use of databases and uses PULCON (pulse length-based concentration determination) quantitative NMR to obtain results that are significantly more accurate and reproducible than those obtained by CPMG (CarrePurcell eMeiboomeGill) sequence or post-processing filtering approaches. Three practical applications of the method highlight its flexibility under different cell culture conditions. We identified and quantified (i) metabolic differences between genetically engineered human cell lines, (ii) alterations in cellular metabolism induced by differentiation of mouse myoblasts into myotubes, and (iii) metabolic changes caused by activation of neurotransmitter receptors in mouse myoblasts. Thus, the new protocol offers an easily implementable, efficient, and versatile tool for the investigation of cellular metabolism and signal transduction.© 2016 Elsevier Inc. All rights reserved.Interest in quantitative nuclear magnetic resonance (q-NMR) analysis has steadily increased since the seminal works of Jungnickel and Forbes [1] and Hollis [2], owing to the unique ability of this methodological approach to quantify, at the same time, a wide range of structurally diverse biological substances with high throughput and automation [3]. Compared with other analytical techniques, q-NMR does not require chromatographic separation, generates signals that are directly proportional to the number of NMR-active nuclei in the targeted analyte [4,5], and offers a high degree of assay reproducibility [6] along with reduced uncertainty [7,8]. Moreover, advances in hardware developmentdsuch as the introduction of high-field magnets and cryogenic probesdhave progressively lowered the limit of detection (LOD) for analytes, thereby improving the overall sensitivity of the technique [4].The PULCON (pulse length-based concentration determination) procedure [9], one of the most promising methods for q-NMR [4], finds its mathematical and physical foundation in the principle of reciprocity [10e12]. This principle states that the strength of a signal is inversely proportional to the 90 pulse length in the active volume of the NMR tube. The PULCON method can be implemented in all types of commercial NMR spectrometers and requires only one Abbreviations used: q-NMR, quantitative nuclear magnetic resonance; LOD, limit of detection; PULCON, pulse length-based concentration determination; CPMG,

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“…Supernatants were used as total lysates. Lysates (50 -100 g of protein) were incubated in AC buffer (100 mM sodium phosphate, 0.1% Nonidet P-40, 150 mM NaCl, 3 mM DTT, 100 mM sodium citrate, pH 4.5) with 50 M N-lauroyl ceramide (Nu-Chek Prep, Elysian, MN) as substrate, reactions were carried on for 1 h at 37°C, and the reaction product was extracted as described (11,12). Blank samples without protein extract or without substrate were used as negative controls.…”

Section: Ac Assay In Total Lysatementioning

confidence: 99%

Acid Ceramidase in Melanoma

Realini

Palese

Pizzirani

et al. 2016

Journal of Biological Chemistry

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Acid ceramidase (AC) is a lysosomal cysteine amidase that controls sphingolipid signaling by lowering the levels of ceramides and concomitantly increasing those of sphingosine and its bioactive metabolite, sphingosine 1-phosphate. In the present study, we evaluated the role of AC-regulated sphingolipid signaling in melanoma. We found that AC expression is markedly elevated in normal human melanocytes and proliferative melanoma cell lines, compared with other skin cells (keratinocytes and fibroblasts) and non-melanoma cancer cells. High AC expression was also observed in biopsies from human subjects with Stage II melanoma. Immunofluorescence studies revealed that the subcellular localization of AC differs between melanocytes (where it is found in both cytosol and nucleus) and melanoma cells (where it is primarily localized to cytosol). In addition to having high AC levels, melanoma cells generate lower amounts of ceramides than normal melanocytes do. This downregulation in ceramide production appears to result from suppression of the de novo biosynthesis pathway. To test whether AC might contribute to melanoma cell proliferation, we blocked AC activity using a new potent (IC 50 ‫؍‬ 12 nM) and stable inhibitor. AC inhibition increased cellular ceramide levels, decreased sphingosine 1-phosphate levels, and acted synergistically with several, albeit not all, antitumoral agents. The results suggest that AC-controlled sphingolipid metabolism may play an important role in the control of melanoma proliferation.Ceramides are a class of bioactive lipids that regulate senescence, apoptosis, and autophagy (1). They comprise more than 200 chemically and functionally distinct molecules and are produced through either de novo biosynthesis or cleavage of preformed sphingolipid precursors in membranes (2-4). They are degraded by the action of five distinct ceramidases, which differ in sequence, structure, subcellular localization, and preferred substrates (5). Among them, acid ceramidase (AC) 2 (also known as N-acetylsphingosine amidohydrolase, ASAH1) is especially relevant due to its possible roles in cancer progression and chemoresistance (6). AC is a lysosomal cysteine amidase that catalyzes the hydrolysis of ceramide into sphingosine and fatty acid with a preference for unsaturated ceramides with 6 -16-carbon acyl chains (5). Its activation is associated with decreased cellular levels of these medium-chain ceramides, which promote senescence and apoptosis, and increased levels of sphingosine 1-phosphate (S1P), which stimulates cell proliferation and cancer cell migration (7). A bioinformatic survey of the National Cancer Institute (NCI) gene expression database conducted in 2003 identified AC as a potential candidate for the development of new biomarkers for the prognosis of melanoma (8). Supporting this possibility, subsequent studies showed that serum of melanoma patients contains AC autoantibodies (9). Moreover, experiments with melanoma cell lines in cultures have demonstrated that dacarbazine, a standard of care for the...

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Benzoxazolone Carboxamides: Potent and Systemically Active Inhibitors of Intracellular Acid Ceramidase

Cited by 16 publications

References 29 publications

Regulation of hepatic insulin signaling and glucose homeostasis by sphingosine kinase 2

Regulation of hepatic insulin signaling and glucose homeostasis by sphingosine kinase 2

A simple and accurate protocol for absolute polar metabolite quantification in cell cultures using quantitative nuclear magnetic resonance

Acid Ceramidase in Melanoma

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