Approaches for probing and evaluating mammalian sphingolipid metabolism.

Sphingolipid metabolism plays a critical role in regulating processes that control cellular fate. This dynamic pathway can generate and degrade the central players: ceramide, sphingosine and sphingosine-1-phosphate in almost any membrane in the cell, adding an unexpected level of complexity in deciphering signaling events. While in vitro assays have been developed for most enzymes in SL metabolism, these assays are setup for optimal activity conditions and can fail to take into account regulatory components such as compartmentalization, substrate limitations, and binding partners that can affect cellular enzymatic activity. Therefore, many in-cell assays have been developed to derive results that are authentic to the cellular situation which may give context to alteration in SL mass. This review will discuss approaches for utilizing probes for mammalian in-cell assays to interrogate most enzymatic steps central to SL metabolism. The use of inhibitors in conjunction with these probes can verify the specificity of cellular assays as well as provide valuable insight into flux in the SL network. The use of inhibitors specific to each of the central sphingolipid enzymes are also discussed to assist researchers in further interrogation of these pathways.