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A Hydroxychalcone Derived from Cinnamon Functions as a Mimetic for Insulin in 3T3-L1 Adipocytes

Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, IA 50011 (K.J.J.-T., D.J.G.)
Human Nutrition Research Center, ARS, USDA, Beltsville, MD 20705 (R.A.A.)

View larger version (53K): [in a new window]   Fig. 1. Tyrosine phosphorylation of the insulin receptor in 3T3-L1 adipocyte cells. 3T3-L1 adipocytes were stimulated with various concentrations of insulin or methylhydroxychalcone polymer (MHCP) for 30 minutes, washed and lysed. The insulin receptor ß subunit was immunoprecipitated, separated by SDS-PAGE and transferred to PVDF membrane. The receptor was probed with antiphosphotyrosine antibodies and detection was with chemiluminescence.

View larger version (21K): [in a new window]   Fig. 2. Effect of MHCP or insulin on glucose uptake stimulation. 3T3-L1 adipocytes were treated with insulin (100 nM), MHCP (0.1 mg/mL) or insulin + MHCP for 30 minutes. The uptake of 2-deoxy-D-[1,2-3H]glucose was measured in cell lysates at 10, 30 and 60 minutes. Data are expressed as the counts obtained in the treatment divided by the counts from the control. The uptake curves represent the mean ± standard deviation of triplicate samples from three experiments.

View larger version (53K): [in a new window]   Fig. 3. Effect of wortmannin treatment on the stimulation of glycogen production in 3T3-L1 adipocytes. 3T3-L1 adipocytes were treated with insulin (100 nM), MHCP (0.1 mg/mL) or insulin + MHCP in combination with various concentrations of wortmannin for 30 minutes and washed. The incorporation of D-[14C]glucose into the glycogen pools (without wortmannin present) was allowed for 60 minutes. The radioactivity incorporated into the glycogen was measured in the glycogen precipitate. The data represent the mean ± standard deviation of triplicate samples from three experiments.

View larger version (48K): [in a new window]   Fig. 4. Effect of wortmannin on the stimulation of glycogen production in 3T3-L1 adipocytes. 3T3-L1 adipocytes were treated with various concentrations of wortmannin in the presence of insulin (100 nM), MHCP (0.1 mg/mL) or insulin + MHCP for 30 minutes. The incorporation of D-[14C]glucose into the glycogen pools in the presence of wortmannin was allowed for 60 minutes. The radioactivity incorporated into the glycogen was measured in the glycogen precipitate. The data represent the mean ± standard deviation of triplicate samples from two experiments.

View larger version (44K): [in a new window]   Fig. 5. Effect of LY294002 on the stimulation of glycogen production in 3T3-L1 adipocytes. 3T3-L1 adipocytes were treated with various concentrations of LY294002 in the presence of insulin (100 nM), MHCP (0.1 mg/mL) or insulin + MHCP for 30 minutes. The incorporation of D-[14C]glucose into the glycogen pools in the presence of LY294002 was allowed for 60 minutes. The radioactivity incorporated into the glycogen was measured in the glycogen precipitate. The data represent the mean ± standard deviation of triplicate samples from three experiments.

View larger version (53K): [in a new window]   Fig. 6. Effect of MHCP or insulin on glycogen synthase activity. 3T3-L1 adipocytes were treated with insulin (100 nM), MHCP (0.1 mg/mL) or insulin + MHCP for 30 minutes, washed and the cell supernate obtained. Glycogen synthase assays were performed using uridine diphosphate D-[14C]glucose in the presence of either 0.1 mM glucose-6-phosphate (G6P) or 10 mM G6P and the radioactivity incorporated into the glycogen precipitant was measured. The low G6P/high G6P ratio was determined and the ratio from the treatment was divided by the ratio obtained from control cells. The data represent the mean ± standard deviation of triplicate samples from three experiments.

View larger version (71K): [in a new window]   Fig. 7. Effect of MHCP or insulin on glycogen synthase kinase3ß activity. 3T3-L1 adipocytes were treated with insulin (100 nM), MHCP (0.1 mg/mL) or insulin + MHCP for 30 minutes, washed and the cell supernate obtained. GSK-3ß assays were performed to measure the incorporation of 32P into the phosphoglycogen synthase peptide 2 (Upstate Biotechnologies). The level of incorporation of the control cells was set at 100% and the other values were set in comparison. The data represent the mean ± standard deviation of triplicate samples from three experiments.