Ceramide production in response to acute TNF-α stimulation in FVB mice. Representative images (a) and quantitative results (b) of cardiac-tissue ceramide production. Detectable ceramide mass measured by DAG-kinase assay was significantly greater in male cardiac tissue than in female in the presence of both TNF-α and NOE. Although treatment with TNF-α or NOE alone resulted in significant increase in ceramide production, ceramide mass content was not significantly different between sexes in those groups. Representative images (c) and quantitative results (d) of liver-tissue ceramide production. By contrast to cardiac tissues, additional treatment with NOE plus TNF-α did not result in a significant difference in terms of ceramide production, although the treatment stimulated a substantial amount of ceramide production in both sexes. (e) Dose response of cardiac-tissue ceramide production in the presence of both TNF-α and NOE. Male hearts produced significantly more ceramide in response to both low (200 U/mL) and high (1,000 U/mL) doses of TNF-α. (f) Standard curve of DAG-kinase assay. Note that both samples with low- (A: –TNF-α, –NOE) and high- (B: +TNF-α, +NOE) ceramide content fall within the linear range of the assay. (g) Correlation between total-protein and phospholipid content of the samples. A strong positive correlation was observed between tissue-protein content and phospholipid content (P < 0.005). Ceramide production was expressed as the percentage of control (–TNF-α, –NOE) values, set at 100%. Values are mean ± SD. ^AP < 0.01 vs. control. ^BP < 0.05 vs. female (+TNF-α, +NOE). ^CP < 0.01.