Troglitazone prevents and reverses dexamethasone induced insulin resistance on glycogen synthesis in 3T3 adipocytes

Abstract

Troglitazone lowers blood glucose levels in Type II diabetic patients. To evaluate the insulin sensitizing action of troglitazone on glycogen synthesis we have used dexamethasone-treated 3T3 adipocytes as an in vitro model. Differentiated 3T3 adipocytes were incubated with 100 nM dexamethasone for 6 days. Troglitazone (1.0 microM) or metformin (1.0 mM) with or without 200 nM insulin was added during the last 4 days. At the end, insulin (100 nM) stimulated glycogen synthesis was determined using (14)C-glucose. Dexamethasone caused a 50% reduction in glycogen synthesis. Troglitazone caused an approximately 3 fold increase in glycogen synthesis from 43.9+/-3.4 to 120+/-16.2 nmols h(-1). Under identical conditions metformin had no significant effect. When cells were incubated with troglitazone and dexamethasone simultaneously for 6 days, troglitazone but not metformin completely prevented dexamethasone-induced insulin resistance. RU 486 (1.0 microM) also completely prevented the insulin resistance. Chronic incubation with dexamethasone and insulin resulted in a 73% reduction in glycogen synthesis. In these adipocytes, troglitazone was partially active with glycogen synthesis rising from 23.1+/-3.0 to 44.4+/-4.5 nmol h(-1), P<0.01 while metformin was inactive. Troglitazone stimulated 2-deoxyglucose uptake by 2 - 3 fold in dexamethasone-treated adipocytes. Metformin also increased glucose uptake significantly. Troglitazone did not affect insulin binding while a 2 fold increase was observed in normal adipocytes where it exhibited a modest effect. Since the effect of troglitazone was greater in dexamethasone-treated adipocytes, troglitazone is likely to act by preventing dexamethasone-induced alterations which may include (i) binding to glucocorticoid receptor and (ii) effect on glucose uptake. These data demonstrate the direct insulin sensitizing action of troglitazone on glycogen synthesis and suggest a pharmacological profile different from metformin.

Figure 1

Effect of dexamethasone on glycogen synthesis in 3T3 adipocytes. Fully differentiated 3T3-adipocytes were incubated for 3 days with 100 nM dexamethasone or vehicle and glycogen synthesis was studied using ¹⁴C-glucose in the presence of 0–100 nM porcine insulin. Data are mean±s.e.mean of n=4 at each insulin concentration. ^*P<0.05 compared to vehicle treatment. Data are presented on a per dish basis. Each dish had 1–2×10⁶ cells. Basal glycogen synthesis of vehicle treated cells corresponds to 13.5±1.5 nmols h⁻¹ and maximal insulin stimulated glycogen synthesis corresponds to 99.3±15.3 nmols h⁻¹.

Figure 2

(a) Dose response of troglitazone on dexamethasone treated adipocytes. 3T3 adipocytes were incubated with 100 nM dexamethasone for 2 days. After 2 days troglitazone 0–10 μM and dexamethasone, 100 nM were added and incubated for 4 days. At the end of 6 days, cells were removed and glycogen synthesis from ¹⁴C-glucose was assayed in the presence of 100 nM insulin. Data are mean±s.e.mean of n=4, ^**P<0.02, ^***P<0.001 compared to dexamethasone-treated adipocytes. (b) Effect of troglitazone and metformin on dexamethasone-treated adipocytes. 3T3 adipocytes were incubated with or without 100 nM dexamethasone for 2 days. After 2 days troglitazone (1.0 μM) or metformin (1.0 mM) was added to dishes incubated with dexamethasone along with 100 nM dexamethasone for the next 4 days. At the end of 6 days, the dishes were removed and glycogen synthesis was studied in the presence of 100 nM insulin. Data are mean±s.e.mean, n=4, ^***P<0.001 compared to dexamethasone-treated cells. (c) Effect of troglitazone and metformin incubated simultaneously with dexamethasone. 3T3 adipocytes were incubated with troglitazone (1.0 μM) or metformin (1.0 mM) or vehicle along with 100 nM dexamethasone for 6 days. At the end of 6 days glycogen synthesis was studied in the presence of 100 nM insulin. Data are mean±s.e.mean, n=8 for troglitazone and n=6 for other groups. ^**P<0.01 compared to dexamethasone-treated cells.

Figure 3

Effect of troglitazone or metformin on 3T3 adipocytes incubated with dexamethasone and insulin. 3T3 adipocytes were incubated for 2 days with 100 nM dexamethasone. After 2 days, troglitazone (1.0 μM) or metformin (1.0 mM) was added and incubated along with 200 nM insulin and 100 nM dexamethasone for a further 4 days. At the end of 6 days, cells were removed and glycogen synthesis was assayed in the presence of 100 nM insulin. Data are mean±s.e.mean n=4, ^*P<0.05 versus dexamethasone alone ^**P<0.01 versus cells treated with chronic insulin and dexamethasone.

Figure 4

Effect of RU 486 on insulin dose response on glycogen synthesis in dexamethasone treated cells. 3T3 adipocytes were incubated with 100 nM dexamethasone or vehicle or 100 nM dexamethasone and 1.0 μM RU 486 for 72 h. After 72 h, cells were removed and glycogen synthesis was assayed. Data are mean±s.e.mean for n=4, ^*P<0.05 and ^**P<0.02 for dexamethasone-treated cells compared to vehicle treatment.

Figure 5

Effect of troglitazone on normal 3T3 adipocytes. 3T3 adipocytes were treated with troglitazone (1.0 μM), or metformin (1.0 mM) or vehicle for 6 days. At the end of 6 days, the cells were removed and glycogen synthesis was assayed in the presence of 100 nM insulin. Data are mean±s.e.mean for n=6, ^*P<0.05 for troglitazone treated cells compared to vehicle treatment.

Figure 6

Effect of troglitazone or metformin on deoxyglucose uptake. 3T3 adipocytes were incubated with 100 nM dexamethasone for 2 days. On day 3, the cells were incubated with troglitazone (1.0 μM) in the presence or absence of 200 nM insulin, or metformin (1.0 mM) and 100 nM dexamethasone for the next 4 days. At the end of 6 days, the cells were removed and ¹⁴C-deoxyglucose uptake was measured in the presence of 25 nM porcine insulin for 20 min. Data are mean±s.e.mean of n=8. ^**P<0.02 for metformin compared to dexamethasone-treated cells. ^***P<0.001 for troglitazone treatments comparing cells incubated with dexamethasone and insulin or dexamethasone alone.