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Gliclazide increases insulin receptor tyrosine phosphorylation but not p38 phosphorylation in insulin-resistant skeletal muscle cells

Naresh Kumar and Chinmoy S. Dey^*

Signal Transduction Research Laboratory, Department of Biotechnology, National Institute of Pharmaceutical Education and Research, Punjab, India

View larger version (33K): [in a new window]   Fig. 1. Effect of the chronic presence of insulin and gliclazide on tyrosine phosphorylation of insulin receptor ß (IR-ß) in C2C12 myotubes. Gliclazide (2 mmol l-1) was added during the last day of differentiation for 24 h to C2C12 cells differentiated in the absence (MF) or in the chronic presence (MFI) of insulin. The cells were then stimulated with 100 nmol l-1 insulin for 5 min and lysed. Cell lysate was immunoprecipitated (IP) with antibodies against IR-ß and western immunoblotted (IB) with anti-phosphotyrosine (pTyr) antibody (A). The blots were stripped and reprobed with IR-ß (B). Experiments were repeated three times and representative blots are shown. Phosphorylation levels of IR (C) were quantified by densitometry and expressed relative to MF (control) samples. Error bars represent the S.E.M. of three independent experiments (*P<0.05).

View larger version (29K): [in a new window]   Fig. 2. Effect of the chronic presence of insulin and gliclazide on tyrosine phosphorylation of insulin receptor substrate 1 (IRS-1) in C2C12 myotubes. Samples were treated as described in Fig. 1. Cell lysate was immunoprecipitated (IP) with antibodies against IRS-1 and western immunoblotted (IB) with anti-phosphotyrosine (pTyr) antibody (A). The blots were stripped and reprobed with IRS-1 (B). Experiments were repeated three times and representative blots are shown. Phosphorylation levels of IRS-1 (C) were quantified by densitometry and expressed relative to MF (controls, differentiated in the absence of insulin) samples. Error bars represent the S.E.M. of three independent experiments (**P<0.01). MFI, cells differentiated in the chronic presence of insulin.

View larger version (21K): [in a new window]   Fig. 3. Effect of the chronic presence of insulin and gliclazide on phosphatidylinositol 3-kinase (PI 3-kinase) activity in C2C12 myotubes. C2C12 cells, differentiated in the absence (MF) or in the chronic presence (MFI) of insulin, were washed with Krebs-Ringer phosphate (KRP) buffer as described in Materials and methods, followed by stimulation with insulin (100 nmol l-1) for 10 min. PI 3-kinase activity in anti-IRS-1 (insulin receptor substrate 1) immunoprecipitates was measured as described under Materials and methods. Cells were treated with 2 mmol l-1 gliclazide during the last 24 h of differentiation. A representative autoradiogram for three independent experiments is shown (A). The relative density of phosphatidylinositol (3,4,5)-trisphosphate [PtdIns(3,4,5)P3] spots (B) was quantified by densitometry and expressed relative to control samples (unstimulated insulin samples). Error bars represent the S.E.M. of three independent experiments (*P<0.05).

View larger version (16K): [in a new window]   Fig. 4. Effect of the chronic presence of insulin and gliclazide on 2-deoxyglucose (2-DOG) uptake in C2C12 myotubes. C2C12 cells, differentiated in the absence (MF) or in the chronic presence (MFI) of insulin, were washed with Krebs-Ringer phosphate (KRP) buffer as described in Materials and methods, followed by stimulation with insulin (100 nmol l-1) for 15 min. [3H]2-DOG uptake was measured as described in Materials and methods. Cells were treated with 2 mmol l-1 gliclazide during the last 24 h of differentiation. Error bars represent the S.E.M. of four independent experiments (*P<0.05).

View larger version (39K): [in a new window]   Fig. 5. Effect of the chronic presence of insulin and gliclazide on extracellular-signal regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 activation. C2C12 cells, differentiated in the absence (MF) or in the chronic presence (MFI) of insulin, were washed with Krebs-Ringer phosphate (KRP) buffer as described in Materials and methods, followed by stimulation with insulin (100 nmol l-1) for 5 min. Lysates were prepared from cells treated with 2 mmol l-1 gliclazide during the last 24 h of differentiation. Protein was resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotted with phospho-specific ERK (pERK) (A), phospho-specific JNK (pJNK) (D), phospho-specific p38 (pp38) (G), ERK (B), JNK (E), p38 (H) antibodies. Phospho-specific blots are representative of experiments performed three times. Error bars of quantified data for pERK (C), pJNK (F) and pp38 (I) represent the S.E.M. of three independent experiments.

View larger version (30K): [in a new window]   Fig. 6. Effect of SB203580 on 2-deoxyglucose (2-DOG) uptake and p38 activation. C2C12 cells, differentiated in the absence (MF) or in the chronic presence (MFI) of insulin, were incubated in Krebs-Ringer phosphate (KRP) buffer for 30 min followed by another incubation of 30 min in the presence of 10 µmol l-1 SB203580. Cells were stimulated with 100 nmol l-1 insulin in the presence of SB203580, followed by 2-DOG uptake for 10 min (A) or immunoblot analysis of p38 activation (B). Cells were treated with 2 mmol l-1 gliclazide during the last 24 h of differentiation. Error bars represent the S.E.M. of three independent experiments.

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