Serum cholesterol selectively regulates glucocorticoid sensitivity through activation of JNK

Glucocorticoids (Gc) are potent anti-inflammatory agents with wide clinical application. We have previously shown that increased serum concentration significantly attenuates regulation of a simple Gc-responsive reporter. We now find that glucocorticoid receptor (GR) regulation of some endogenous transactivated but not transrepressed genes is impaired, suggesting template specificity. Serum did not directly affect GR expression, activity or trafficking, implicating GR crosstalk with other signalling pathways. Indeed, a JNK inhibitor completely abolished the serum effect. We identified the Gc modulating serum component as cholesterol. Cholesterol loading mimicked the serum effect, which was readily reversed by JNK inhibition. Chelation of serum cholesterol with methyl-β-cyclodextrin or inhibition of cellular cholesterol synthesis with simvastatin potentiated the Gc response. To explore the effectin vivowe usedApoE−/−mice, a model of hypercholesterolaemia. Consistent with ourin vitrostudies, we find no impact of elevated cholesterol on the expression of GR, or on the hypothalamic–pituitary–adrenal axis, measured by dexamethasone suppression test. Instead we find selective Gc resistance on some hepatic target genes inApoE−/−mice. Therefore, we have discovered an unexpected role for cholesterol as a selective modulator of Gc actionin vivo. Taken together these findings reveal a new environmental constraint on Gc action with relevance to both inflammation and cancer.

Direct enzyme-linked immunosorbent assay and a radioimmunoassay for melatonin compared

A novel commercially available ELISA for direct measurement of melatonin concentration in serum was evaluated and compared with an RIA routinely used in our laboratory. The direct ELISA is technically simpler, requires a smaller sample volume (0.1 mL), and compares well with RIA in terms of stability of the calibration curve and intra- and interassay CVs. Correlation with RIA measurements is, however, suboptimal (y = 0.39x + 56; r = 0.65, P < 0.001; n = 138), which may be due to a serum effect, as evidenced by dilution studies. Furthermore, the detection range of the ELISA does not cover the physiological daytime melatonin concentrations in humans. Adding an extraction and 10-fold concentration step shifted the detection range of the ELISA to include low physiological concentrations as well. Correlation with RIA measurements also improved significantly (y = 0.97x-23; r = 0.95, P < 0.001; n = 105), probably due to removal of the serum effect. Although extraction increases the required sample volume (1.5 mL), work load, and procedure time, this step is necessary for the ELISA to compete successfully with RIA.