Modulation by phenobarbital of the differentiation of 3T3 preadipocytes

Abstract

The effect of phenobarbital upon the differentiation of two preadipocyte cell lines, 3T3 F442A and 3T3 L-1, was examined by measuring the synthesis and secretion of lipoprotein lipase. Extracellular enzyme was measured by treating intact cells with heparin, and the intracellular enzyme was subsequently assayed in cell homogenates. When confluent cultures of 3T3 F442A cells were treated with insulin, the cells underwent differentiation as indicated by increased activity of lipoprotein lipase within 6 days, followed in turn by increased levels of protein and triglyceride. Addition of phenobarbital with insulin enhanced total lipoprotein lipase, protein, and triglyceride content. The activity of lipoprotein lipase accumulated in the heparin-releasable fraction during differentiation was increased 2- to 3-fold and the intracellular enzyme was enhanced 15- to 20-fold by the addition of phenobarbital. The ability of phenobarbital to modulate differentiation was dependent upon the time of addition. When added early in the postconfluent period, there was a greater increase in lipoprotein lipase activity than when the drug was added at later times. Phenobarbital also stimulated lipoprotein lipase in differentiating 3T3 L-1 cells in the presence of insulin, although lipoprotein lipase activity was moderately enhanced by phenobarbital alone in these cells. These results suggest that phenobarbital may affect the conversion of adipoblasts into preadipocytes and thereby increase the proportion of cells susceptible to the differentiating stimulus.

Effect of Alcohol on Lipoprotein Metabolism

“…After vigorous mixing and centrifugation, the amount of oleate present in the upper aqueous layer was measured by liquid scintillation counting. Hormone-sensitive lipase does not account for more than 3.5 % of the hydrolysis of triolein by adipose tis sue under these assay conditions [22]. HTGL has not been demonstrated in heart muscle, therefore the lipolytic activity in this tissue in the presence of serum activator (apo C-II) was assumed to represent LPL.…”

“…Viability of freshly prepared hepatocytes was greater ( 1 h), and fresh medium containing heparin (2 U/ml) was added, from which samples were taken for HTGL assay over the next 24 h. Each time point is the mean HTGL activity accumulated in the medium of 3-4 dishes. Error bars show the range of values for each HTGL and LPL Activities LPL and HTGL activities were measured as the release of oleate from a gum arabic-stabilized emul sion of triolein as described previously [21,22]. Briefly, [14C]-triolein (New England Nuclear, Toron to, Ont., Canada) was emulsified in 0.4 mol/1 Tris-HC1 buffer (pH 8.4) containing 0.2 mol/1 NaCl, 25 mg/ml fatty acid-free bovine serum albumin (Sig ma, St. Louis, Mo., USA) and 12.5 mg/ml gum pose, cells after 3 days in culture were treated with heparin medium to remove extracellular HTGL which was discarded, and then 8 ml of fresh medium containing heparin (2 U/ml) was added to each plate.…”