Effects of different differentiation methods on fat accumulation, leptin expression, and secretion in day 14 3T3-L1 adipocytes. (A) Oil red O staining in fully differentiated adipocytes. (B) Leptin mRNA levels, relative to cyclophilin A mRNA levels were determined using quantitative real-time PCR. Each bar is the mean±SE of 5 independent experiments normalized to the differentiation with Ibmx, Dex, Ins, and Tzd. (C) Representative experiment of leptin accumulation into the medium over 48 h normalized to total protein. Data of three wells of a 6-well culture dish were combined for each condition. The experiment was repeated over 8 times all with equivalent data.

Leptin secretion from 3T3-L1 adipocytes is independent of protein synthesis and requires trafficking through the Golgi apparatus. (A) Effect of cyclohexamide (CHX) on insulin-induced leptin secretion over 2 h. Cells were pretreated with 10 μg/ml cyclohexamide for 30 min before stimulated with 170 nM insulin for 2 h. Each bar is a mean± SE of 3 independent experiments normalized to the insulin values. (B) Effect of Brefeldin A (BFA) on short-term insulin-regulated leptin secretion. Cells were treated with 5 μg/ml Brefeldin A for 30 min before 15-min insulin stimulation. Each bar is a mean ± SE of 6 independent experiments normalized to the insulin-stimulated state. *p = 0.001.

Acute insulin-induced leptin secretion from adipocytes differentiated with Ibmx/Dex/Ins plus PPARγ agonist. (A) Scheme of a designed secretion assay with short-term insulin stimulation (170 nM). 3T3-L1 fibroblasts were differentiated with Ibmx/Dex/Ins plus Tzd cocktail and cultured until fully differentiated (day 14). On the night before the experiment medium was changed to minimal amounts (0.5 ml) in order to allow leptin to accumulate for 15 h overnight. In the morning, an overnight point was taken as a starting concentration for leptin secretion and cells were stimulated with 170 nM insulin for 2 h. Medium was collected from 3 wells of a 6-well plate, concentrated using Millipore filter units and leptin content was assayed using a Quantikine Elisa kit. Accumulated leptin was normalized to total amount of protein from each well. (B) Representative experiment of insulin-induced leptin secretion over 15 min and 2 h from adipocytes. The experiment was repeated over 10 times all with equivalent data.

Insulin-induced leptin secretion from 3T3-L1 adipocytes is independent of the PI 3-kinase signaling pathway and of glucose metabolism. (A) 3T3-L1 cells were cultured and differentiated with Ibmx/Dex/Ins plus Tzd as described in Fig. 2 until day 15, at which stage the medium was changed to either standard DMEM containing 4.5 g/l glucose or glucose-free DMEM both supplemented with 10% FBS. After 2 h with or without 170 nM insulin the medium was collected, concentrated and assayed for secreted leptin. Data of three wells of a 6-well culture dish were combined for each condition. The experiment was repeated 4 times all with equivalent data. (B) Wortmannin (WT) does not block insulin-induced secretion of leptin over 2 h. Cells were treated with 100 nM or 1 μM wortmannin for 30 min prior to 2-hour insulin stimulation. Each bar is a mean±SE of 5 independent experiments normalized to the insulin-stimulated state values. (C) 3T3-L1 adipocytes were treated with or without 100 nM or 1 μM wortmannin for 30 min before insulin stimulation for 2 h. Cell lysates were analyzed in immunoblots by using noted antibodies.