On the nature of ceramide-mitochondria interactions - Dissection using comprehensive mitochondrial phenotyping

Abstract

Sphingolipids are a unique class of lipids owing to their non-glycerol-containing backbone, ceramide, that is constructed from a long-chain aliphatic amino alcohol, sphinganine, to which a fatty acid is attached via an amide bond. Ceramide plays a star role in the initiation of apoptosis by virtue of its interactions with mitochondria, a control point for a downstream array of signaling cascades culminating in apoptosis. Many pathways converge on mitochondria to elicit mitochondrial outer membrane permeabilization (MOMP), a step that corrupts bioenergetic service. Although much is known regarding ceramides interaction with mitochondria and the ensuing cell signal transduction cascades, how ceramide impacts the elements of mitochondrial bioenergetic function is poorly understood. The objective of this review is to introduce the reader to sphingolipid metabolism, present a snapshot of mitochondrial respiration, elaborate on ceramides convergence on mitochondria and the upstream players that collaborate to elicit MOMP, and introduce a mitochondrial phenotyping platform that can be of utility in dissecting the fine-points of ceramide impact on cellular bioenergetics.

Keywords: Bioenergetics; Cancer; Ceramide; Mitochondria; Sphingolipids.

Fig. 1.. Biosynthesis of ceramide by the de novo pathway.

Ceramide is composed of a sphingolipid base, sphinganine, joined by an amide bond to a fatty acid. Ceramide is synthesized in the endoplasmic reticulum. The de novo steps begin with the condensation of serine and palmitoyl-CoA, catalyzed by serine palmitoyltransferase. The product, 3-ketosphinganine, contains 18 carbons and is reduced to sphinganine by 3-ketosphinganine reductase. The next step generates ceramides saturated precursor, dihydroceramide, via the action of dihydroceramide synthase, of which there are several isoforms that ultimately give rise to a multitude of molecular species of ceramide with distinct roles. Finally, although dihydroceramide is nearly identical in structure to ceramide, it lacks the 4, 5-trans double bond, which is inserted by dihydroceramide desaturase to form ceramide.

Fig. 2.. Cellular fate of ceramide.

Ceramide can be produced by the action of specialized phospholipases, known as sphingomyelinases. Sphingomyelinases, which are characterized according to their optimum pH and subcellular locations, cleave sphingomyelin at the phosphodiester bond that is proximal to ceramide, producing ceramide and choline phosphate. Ceramide can also be generated by the action of ceramide 1-phosphate phosphatase (C1-P phosphatase) and via glucosylcerebrosidase (Gluc-ase). Once produced, ceramide can be hydrolyzed by ceramidase, glycosylated by glucosylceramide synthase (GCS), or phosphorylated by ceramide kinase producing ceramide 1-phosphate (C1-P). Strategic points in de novo synthesis and in subsequent ceramide metabolism can be activated or inhibited, providing useful avenues for studying ceramide-regulated events and for controlling cell fate. Enzyme inhibitors and P-glycoprotein (P-gp) antagonists are often used to amplify the induction of cell death by ceramide. Enzymes that “remove” ceramide either by destructive, ceramidase, or constructive metabolism, glucosylceramide synthase (GCS), can contribute to cancer cell growth.

Fig. 3.. Mitochondrial Energy Transduction.

Mitochondrial energy transfer mediated by the dehydrogenase network, ETS, and the phosphorylation system. Mitochondrial flux is depicted as a H⁺ circuit conducted via various ‘OXPHOS’ and ‘Non-OXPHOS’ resistors; ‘?’–predicted ‘Non-OXPHOS’ resistors. Proton motive force (Δp), Complex V (CV), Adenine nucleotide translocase (ANT), Nicotinamide nucleotide transhydrogenase (NNT), mitochondrial calcium uniporter (MCU), Uncoupling protein (UCP).

Fig. 4.. Musicians in the intrinsic pathway of apoptosis—a large ensemble.

The intrinsic mitochondrial pathway of ceramide-assisted apoptosis is largely regulated by caspases and Bcl-2 family members. Crosstalk between signaling members is designated by arrows. Loss of mitochondrial outer membrane permeability (MOMP) subsequently leads to the release of proapoptotic proteins: cytochrome c, apoptosis-inducing factor (AIF), and second mitochondria-derived activator of caspase (SMAC)–direct inhibitor of apoptosis protein (IAP)-binding protein with low PI (DIABLO). GSK3β, glycogen synthase kinase 3β; HRK, harakiri; JNK, JUN N-terminal kinase; PKC, protein kinase C; PP2A, protein phosphatase 2A; TXNIP, thioredoxin interacting protein.

Fig. 5.. Effect of SACLAC exposure on ceramide levels in drug-resistant leukemia cells.

HL-60/dnr cells (resistant to daunorubicin) (800,000/ml dnr-free, RPMI-1640 medium, 10% FBS) were exposed to 10 μM SACLAC (DMSO vehicle) or DMSO (control) for 24 hr. Cells were then harvested by centrifugation, washed three times in PBS, and subjected to lipidomic evaluation by LC/ESI/MS/MS. A. Ceramide molecular species. B. Dihydroceramide molecular species. Viability in SACLAC-treated cells was 80% at harvest. Data are expressed as fold change compared with untreated controls. Data from Kao L-P, et al., J. Lipid Res., 60: 1590–1602, 2019, and reproduced with permission from the publisher.

Fig. 6.. Effect of SACLAC exposure on mitochondrial bioenergetics in drug-resistant leukemia.

HL-60/dnr cells (resistant to daunorubicin) (800,000/ml dnr-free, RPMI-1640 medium, 10% FBS) were exposed to 10 μM SACLAC (DMSO vehicle) or DMSO (control) for 24 hr. Cells were then harvested by centrifugation, washed three times in PBS, and subjected to bioenergetic characterization. For respiration experiments, cells were suspended in a potassium-based respiration buffer, permeabilized with digitonin (10μg/mL), and energized with various carbon substrates and respiratory stimuli/inhibitors. All data were normalized to live cell count. A. Cell viability. B. Respiration under basal conditions, as well as in response to digitonin (Digi, 10μg/mL), pyruvate/malate (Pyr/M, 5mM/1mM), cytochrome c (Cyt C, 10μM), CK clamp (CK 20U/mL; phosphocreatine, PCR, 1mM; ATP, 5mM), octanoyl-carnitine/glutamate/succinate (O/G/S, 0.2mM/5mM/5mM); and multiple PCR additions to titrate ATP free energy (ΔG_ATP) across a physiological span. C. Relationship between oxygen consumption (JO₂) and ATP free energy (ΔG_ATP). Calculation of ΔG_ATP done using the online resource https://dmpio.github.io/bioenergetic-calculators/ck_clamp/. D. Respiration under basal conditions, as well as in response to digitonin (Digi, 10μg/mL), Pyr/M/O/G/S (Multi), cytochrome c (Cyt C, 10μM), and FCCP titration (0.5–3.0μM). Rotenone (0.5μM) and antimycin A (0.5μM) were added at the end to control from any non-mitochondrial respiration. Data are mean ± SEM, N=3/group, *P<0.05.