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Abstract
Background
The target of the treatment of metabolic syndrome and diabetes is an improvement of insulin resistance. D-chiro-inositol (DCI) plays a role in a phospholipid mediating intracellular insulin action. In the previous studies, the urine level of DCI were decreased in the diabetic animal with insulin resistance. Some clinical studies showed that DCI improved a glucose level and HbA1c. Therefore we studied the relationship between DCI and glucose metabolism, especially insulin resistance.
Methods
To investigate the mechanism of DCI affecting the glucose metabolism, we examined the effects of DCI on 2-deoxyglucose uptake, gene expression of adipocytokines and AMPK pathway by using RT-PCR and western blot in 3T3-L1 cells.
Results
Insulin-stimulated 2-deoxyglucose uptake increased in DCI-treated cells by about 1.2-fold (relative to the control) and was inhibited by phosphoinositide 3-kinase (PI3 Kinase) inhibitors (Wortmanin, LY294002) and AMPK inhibitor (STO-609). In Western blot analysis, it didn't show the difference of phosphorylation of Akt and AMPK between DCI-treated group and control in 3T3-L1 cells. However, DCI decreased the gene expression of resistin in 3T3-L1 cells.
Figures and Tables
Fig. 1
Effect of DCI on glucose uptake. DCI-induced glucose uptake is increased and is decreased by PI3 Kinase inhibitor (Wortmannin, Ly294002) and AMPK inhibitor (STO-609), relatively. After a 2 hrs starvation period, 3T3-L1 cells were preincubated with 100 µM DCI for 10 min. 5 mM NaCl was added as control for the osmtic effect. The 3T3-L1 cell were cultured in 10% FBS-DMEM media. In glucose uptake used by 2-deoxyglucose, DCI increased about 1.2 fold relative to control and inhibited by PI3 Kinase inhibitor (Wortmannin, LY294002) and AMPK inhibitor (STO-609).
Fig. 2
Effects of DCI on Akt and AMPK. A. phosphorylation of Akt. B. phosphorylation of AMPK. It didn't show phosphorylation of Akt and AMPK between DCI-treated group and control. The 3T3-L1 cells were cultured in 10% FBS-DMEM media. At 30 minutes after insulin (10 µg/mL) or DCI (10 µM, 100 µM, 1 mM, 10 mM) treatment, total cell lysates were resolved by SDS-PAGE and analyzed by western blot using anti-Akt, anti-phospho (Ser473, Thr308), anti-AMPK, anti-phospho AMPK antobodies. The blot developed by ECL.
Fig. 3
Effects of DCI on adipocytokines. A. Leptin gene expression after 24 hours-treatment. B. Resistin gene expression after 24 hours-treatment. Analysis of variance (ANOVA) was done among the three groups (Vehicle, DCI 100 µM and 1 mM groups). DCI 100 µM or 1 mM decreased significantly (P < 0.05) the gene expression (0.52 ± 0.18 or 0.45 ± 0.28) of resistin in 3T3-L1 cells (n = 4), compared to Vehicle group. DCI 100 µM or 1 mM did not change significantly the gene expression (0.93 ± 0.09 or 1.56 ± 0.49) of leptin in 3T3-L1 cells (n = 4), compared to Vehicle group. Extraction of total RNA and quantitative real-time RT-PCR were performed as described under Materials and Methods. Leptin and resistin gene expression normalized to β-actin mRNA level is relative to untreated control (vehicle) cells.
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