Martin J. Smit

DIFFERENCES IN PROPIONATE-INDUCED INHIBITION OF CHOLESTEROL AND TRIACYLGLYCEROL SYNTHESIS BETWEEN HUMAN AND RAT HEPATOCYTES IN PRIMARY CULTURE

Propionate is a short-chain fatty acid formed in the colon and supposedly involved in the cholesterol-lowering effect of soluble fibre. To explore the underlying mechanism(s) of this fibre action, we have used human hepatocytes in primary culture to study the effects of propionate on hepatic lipid synthesis. Initial experiments with mevalonate and mevinolin, a competitive inhibitor of hydroxymethylglutaryl (HMG)-CoA reductase (EC 1.1.1.88) were performed to evaluate basic regulatory mechanisms in these cells; results were compared with those obtained with rat hepatocytes. Incubation for 24 h with mevalonate caused a similar, concentration-dependent inhibition of [14C]acetate incorporation into cholesterol in human and rat hepatocytes. Likewise, mevinolin (100 mumol/l) inhibited the formation of cholesterol from radiolabelled acetate by about 80% in cells from both species. Propionate inhibited cholesterol as well as triacylglycerol synthesis from [14C]acetate with a similar concentration-dependency in rat hepatocytes. Fifty percent inhibition was obtained at a propionate concentration of only 0.1 mmol/l. This propionate-induced inhibition was not affected by a 100-fold excess of unlabelled acetate. Human hepatocytes were much less susceptible in this respect: propionate concentrations of 10-20 mmol/l were required to obtain similar inhibitory effects in these cells, i.e. values greatly exceeding reported portal propionate concentrations in humans. The results suggest the existence of differences in the regulation of hepatic cholesterol (and triacylglycerol) synthesis between human and rat liver cells. These results do not support the hypothesis that the fibre-induced decrease in plasma cholesterol concentration in man is mediated by a direct effect of propionate on hepatic cholesterol synthesis.

Differential effects of eicosapentaenoic acid on glycerolipid and apolipoprotein B metabolism in primary human hepatocytes compared to HepG2 cells and primary rat hepatocytes

We compared the effects of eicosapentaenoic acid (EPA) and oleic acid (OA) on glycerolipid and apolipoprotein B (apoB) metabolism in primary human hepatocytes, HepG2 cells and primary rat hepatocytes. Cells were incubated for 1 to 5 h with 0.25 mM bovine serum albumin in the absence (control) or presence of 1 mM of EPA or OA. Synthesis and secretion of [3H]glycerolipid were determined after 1 h incubation with [3H]glycerol. Cellular and medium apoB abundance was semi-quantitatively estimated in human cells by Western blotting. The following observations were made. (1) Compared to control, OA induced a 7-fold increase in [3H]triacylglycerol (TG) synthesis in human hepatocytes and a 4-fold increase in rat hepatocytes and HepG2 cells. EPA enhanced [3H]TG synthesis about 2-fold in all three cell types although it stimulated [3H]diacylglycerol (DG) synthesis to an extent (i.e., 2.5- to 5-fold) similar to OA. (2) In contrast to OA, which stimulated VLDL-associated [3H]TG secretion 2.5- to 3-fold in the three cell types relative to control, EPA did not alter [3H]TG secretion in HepG2 and rat hepatocytes and suppressed [3H]TG secretion by 75% in primary human hepatocytes. (3) In primary human hepatocytes, both OA and EPA did not alter cellular apoB abundance but EPA decreased apoB secretion by 44% as compared to control. In contrast, both EPA and OA increased cellular and medium apoB abundance 2- to 2.5-fold in HepG2 cells, although medium apoB tended to be lower in EPA-treated cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Org 214007-0: A Novel Non-Steroidal Selective Glucocorticoid Receptor Modulator with Full Anti-Inflammatory Properties and Improved Therapeutic Index

Glucocorticoids (GCs) such as prednisolone are potent immunosuppressive drugs but suffer from severe adverse effects, including the induction of insulin resistance. Therefore, development of so-called Selective Glucocorticoid Receptor Modulators (SGRM) is highly desirable. Here we describe a non-steroidal Glucocorticoid Receptor (GR)-selective compound (Org 214007-0) with a binding affinity to GR similar to that of prednisolone. Structural modelling of the GR-Org 214007-0 binding site shows disturbance of the loop between helix 11 and helix 12 of GR, confirmed by partial recruitment of the TIF2-3 peptide. Using various cell lines and primary human cells, we show here that Org 214007-0 acts as a partial GC agonist, since it repressed inflammatory genes and was less effective in induction of metabolic genes. More importantly, in vivo studies in mice indicated that Org 214007-0 retained full efficacy in acute inflammation models as well as in a chronic collagen-induced arthritis (CIA) model. Gene expression profiling of muscle tissue derived from arthritic mice showed a partial activity of Org 214007-0 at an equi-efficacious dosage of prednisolone, with an increased ratio in repression versus induction of genes. Finally, in mice Org 214007-0 did not induce elevated fasting glucose nor the shift in glucose/glycogen balance in the liver seen with an equi-efficacious dose of prednisolone. All together, our data demonstrate that Org 214007-0 is a novel SGRMs with an improved therapeutic index compared to prednisolone. This class of SGRMs can contribute to effective anti-inflammatory therapy with a lower risk for metabolic side effects.