Adipose Obese Gene Product: RESULTS

Experiment 1

In the absence of leptin, insulin increased (p < 0.05) thecal cell numbers (Fig. 1), and progesterone (Fig. 2A) and androstenedione (Fig. 2B) production to 1.2-, 2.6-, and 5.4-fold, respectively, of controls. Leptin at 10, 100, and 300 ng/ml increased (p < 0.05) thecal cell numbers by 8%, 10%, and 16%, respectively, above that observed for insulin-treated controls (Fig. 1). In contrast, leptin caused a dose-dependent decrease (p < 0.05) in insulin-induced progesterone (Fig. 2A) and androstenedione (Fig. 2B) production. Insulin-induced increases in progesterone and androstenedione production were almost completely blocked (i.e., > 80% inhibition) by 300 ng/ml of leptin (Fig. 2). The concentrations of leptin necessary to inhibit 50% of the progesterone and androstenedione production (/C50; calculated from inhibition curves that were linearized using a semi-log plot) were 28 ng/ml and <10 ng/ml, respectively.

Experiment 2

After 2 days of treatment, insulin, leptin, or insulin plus leptin had no effect (p > 0.10) on thecal cell viability (Fig. 3). Averaged across all four treatments, cell viability was 94.7 ± 1.2%.

Experiment 3

Both 3 and 30 ng/well of insulin inhibited (p < 0.05) specific [125I]insulin binding by thecal cells (Fig. 4). In contrast, neither 3 nor 30 ng/well of leptin competed for specific thecal cell [125I]insulin binding (Fig. 4). ventolin inhaler

Thecal cells specifically bound 125I-leptin in a time-dependent manner, with maximal binding achieved between 2 and 6 h of incubation at 25°C (Fig. 5). Scatchard analysis of specific binding of 125I-leptin to thecal cells (Fig. 6) revealed that leptin binding was of high affinity, with an estimated dissociation constant (Kd) of 2.32 X 10 10 M, and of low capacity, with an estimated receptor concentration of 1.45 fmol/106 cells.

Figure1Adipose obese gene product
FIG. 1. Effect of leptin on insulin-stimulated thecal cell proliferation (experiment 1). Thecal cells from large (a 8 mm) follicles were cultured for 2 days in the presence of 10% FCS, and then treated in serum-free media with 100 ng/ml of LH, and 0 (open bar) or 100 (hatched bars) ng/ml of insulin for an additional 2 days. Medium was changed every 24 h. During the last 2 days of culture, leptin (0, 10, 30, 100, or 300 ng/ml) was also added to the medium. Values are means of four separate experiments; within each replicate experiment, each treatment was applied in triplicate culture wells. Means without a common superscript differ (p < 0.05).

Fig2Adipose obese gene product
FIG. 2. Effect of leptin on insulin-stimulated progesterone (A) and androstenedione (B) production by thecal cells (experiment 1). Thecal cells from large follicles were cultured as described in Figure 1 legend. During the last 2 days of culture, leptin (0, 10, 30, 100, or 300 ng/ml) was added to the medium with LH and either 0 (open bar) or 100 ng/ml (hatched bar) of insulin. Values are means of four separate experiments; within each replicate experiment, each treatment was applied in triplicate culture wells. Within a panel, means without a common superscript differ (p < 0.05).

Fig3Adipose obese gene product
FIG. 3. Lack of effect of insulin and leptin on thecal cell viability (experiment 2). Thecal cells from large follicles were cultured as described in Figure 1 legend. During the last 2 days of culture, insulin (100 ng/ml) and(or) leptin (300 ng/ml) were added to the medium. Values are means of three separate experiments; within each replicate experiment, each treatment was applied in triplicate culture wells.

FIG. 4. Comparison of leptin and insulin on competing for insulin binding by thecal cells collected from large follicles (experiment 3). Thecal cells were cultured for 3 days in the presence of 10% FCS, and then cells were washed and insulin binding assays were conducted as described in Materials and Methods. Values are means of three separate experiments and are expressed as a percentage of total binding. Within each replicate experiment, each treatment was applied in triplicate culture wells. * Mean differs (p < 0.05) from control.

Fig5Adipose obese gene product
FIG. 5. Time course of specific [l25l] leptin binding sites in bovine thecal cells (experiment 4a). Thecal cells were cultured for 3 days in the presence of 10% FCS, and then cells were washed and leptin binding assays were conducted as described in Materials and Methods. Values are means of three separate experiments; within each replicate experiment, each time-point was derived from duplicate total binding wells and duplicate nonspecific binding wells.

Fig6Adipose obese gene product
FIG. 6. Estimation of the number of leptin receptor sites and dissociation constant in bovine thecal cells (experiment 4b). Thecal ceils were cultured as described in Figure 5. Cells were incubated with increasing amounts of [,25l]leptin in the presence or absence of excess unlabeled leptin (see Materials and Methods) and subjected to Scatchard analysis . Data presented are from one representative experiment.

This entry was posted in Obese and tagged Gene Product, Leptin, Obese, Thecal.