Related Experiment Videos
Nuclear triiodothyronine binding in mononuclear leukocytes in normal subjects and obese patients before and after
Abstract:
We evaluated the maximum binding capacity (MBC) and dissociation constant (Kd) of nuclear T3 receptors (NT3R) in mononuclear cells in normal and obese subjects before and after fasting. Mononuclear cells were isolated by isopycnic centrifugation of 50 ml heparinized blood underlayered with Ficoll-Paque. From 1-5 x 10(6) cells (12-60 microgram DNA) were incubated for 2 h at 37 C in 0.5 ml Ham's F-10 medium with 0.5% bovine serum albumin, 25 mM Hepes buffer (pH 7.4), and six T3 concentrations, each in triplicate (free T3 from 7 x 10(-13) to 5 x 10(-11) and 1.5 x 10(-8) M). Nuclei were isolated from washed cells in sucrose (0.25 M)-Tris (20 mM; pH 7.85)-MgCl2 (1.1 mM) containing 0.5% Triton X-100. Cells took up approximately 5% of the medium T3, and this was not significantly influenced by the T3 concentrations in the medium. About 10% of the cellular T3 was bound to nuclei at 7 x 10(-13) M free T3 in the medium. Nuclear binding in the presence of 1.5 x 10(-8) M free T3 was approximately 20% of that at 7 x 10(-13) M. T4 could compete with T3 for nuclear binding, but it was less than 10% as effective as T3 based on free hormone concentrations. MBC and Kd values of NT3R were the means of two separate determinations on successive days with coefficients of variation of 26% and 28%, respectively. The MBC of NT3R in 9 normal subjects was 2.3 +/- 0.4 (SD) x 10(-15) mol T3/100 microgram DNA, and the apparent Kd was 2.3 +/- 0.5 (SD) x 10(-11) M free T3. For 10 obese subjects, the MBC was 2.5 +/- 0.8 (SD) x 10(-15) mol T3/100 microgram DNA, and the Kd was 2.4 +/- 0.6 (SD) x 10(-11) M free T3. During a 16-day total fast, 7 patients lost 10 +/- 2 kg, and the mean serum T3 decreased by 60%. However, the MBC [2.7 +/- 0.9 vs. 2.4 +/- 0.4 (fasted) x 10(-15) mol/100 microgram DNA] and Kd [2.4 +/- 0.7 vs 2.1 +/- 0.4 (fasted) x 10(-11) M free T3] were not significantly altered. Circulating human mononuclear cells contain limited capacity, high affinity binding sites for T3. We are unable to detect differences in either the MBC or Kd for these sites between lean and obese subjects, nor does fasting alter either of these parameters. If fasting alters NT3R in man, such changes are not detectable in circulating mononuclear cells using these techniques.