Regulation of plasma ceramide levels with fatty acid oversupply: evidence that the liver detects and secretes de novo synthesised ceramide

Abstract

Aims/hypothesis: Plasma ceramide concentrations correlate with insulin sensitivity, inflammation and atherosclerotic risk. We hypothesised that plasma ceramide concentrations are increased in the presence of elevated fatty acid levels and are regulated by increased liver serine C-palmitoyltransferase (SPT) activity.

Methods: Lean humans and rats underwent an acute lipid infusion and plasma ceramide levels were determined. One group of lipid-infused rats was administered myriocin to inhibit SPT activity. Liver SPT activity was determined in lipid-infused rats, and obese, insulin resistant mice. The time and palmitate dose-dependent synthesis of intracellular and secreted ceramide was determined in HepG2 liver cells.

Results: Plasma ceramide levels were increased during lipid infusion in humans and rats, and in obese, insulin-resistant mice. The increase in plasma ceramide was not associated with changes in liver SPT activity, and inhibiting SPT activity by ~50% did not alter plasma ceramide levels in lipid-infused rats. In HepG2 liver cells, palmitate incorporation into extracellular ceramide was both dose- and time-dependent, suggesting the liver cells rapidly secreted the newly synthesised ceramide.

Conclusions/interpretation: Elevated systemic fatty acid availability increased plasma ceramide but this was not associated with changes in hepatic SPT activity, suggesting that liver ceramide synthesis is driven by substrate availability rather than increased SPT activity. This report also provides evidence that the liver is sensitive to the intracellular ceramide concentration, and an increase in liver ceramide secretion may help protect the liver from the deleterious effects of intracellular ceramide accumulation.

Fig. 1

Lipid infusion increases plasma NEFA and ceramide levels. (a, b) plasma NEFA and ceramide levels in humans infused with either saline (control, white) or lipid and heparin (lipid, black) *p<0.05 vs control (by two-way ANOVA); n=7 per group. (c) Linear regression analysis of NEFA and ceramide levels in humans infused with lipid and heparin. R²=0.75, p<0.0001. (d, e) plasma NEFA and ceramide levels in rats infused with either 2.5% glycerol (control, white) or lipid and heparin (lipid, black) for 5 h. Bar represents the duration of infusion. (f) Liver ceramide content, *p<0.05 vs control, ^†p<0.05 main effect of infusion (by two-way ANOVA), n=4–14. (g) Maximal ex vivo liver SPT activity, (h) plasma NEFA, (i) ceramide levels in rats infused with either 2.5% glycerol (control) or lipid and heparin (lipid) for 5 h, administered 100 μg/kg myriocin i.v. 5 min prior to infusion (black) or saline (white). *p<0.05 vs no myriocin; ^†p<0.05 main effect of infusion; n=5–8 per group. Values are mean±SEM

Fig. 2

Dose and time course of incorporation of palmitate or serine into intracellular or extracellular ceramide in HepG2 cells. (a, b) Dose–response for incorporation of palmitate into intracellular or extracellular ceramide. (c, d) Time course of incorporation of palmitate into intracellular or extracellular ceramide. Cells were incubated with 0.5 mmol/l cold palmitate and 0.4 mmol/l cold serine. (e, f) Time course of incorporation of L-serine into intracellular or extracellular ceramide. Cells were incubated with 0.5 mmol/l cold palmitate and 0.4 mmol/l cold L-serine. Linear regression analysis was performed: (b) R²=0.94, p<0.0001, (d) R²=0.81, p<0.0001, (f) R²=0.95, p< 0.0001. Values are mean±SEM. *p<0.05 vs t=0 (by one-way ANOVA), n=3 per group. (g) Schematic diagram of proposed model whereby in models of increased long-chain fatty acid (LCFA) availability, activated LCFA (LCFA-CoA) is converted into ceramide via de novo ceramide synthesis and then packaged into VLDL and LDL, which has been associated with various pathologies