Cholesterol Depletion Blocks Redistribution of Lipid Raft Components and Insulin-Mimetic Signaling by Glimepiride and Phosphoinositolglycans in Rat Adipocytes

Table 2 Effect of CO treatment and withdrawal on pp59 ^Lyn translocation and IRS-1 tyrosine phosphorylation

	pp59^Lyn Translocation to lcDIGs			IRS-1 Tyrosine Phosphorylation
	Control	+CO	+ CO > −CO 180 min	Control	+CO	+CO > −CO 120 min
Basal	1.0 ± 0.2	3.7 ± 0.5	1.5 ± 0.2	1.0 ± 0.1	1.1 ± 0.2	0.9 ± 0.1
Na₃VO₄	1.3 ± 0.3	3.5 ± 0.4	1.4 ± 0.2	10.6 ± 1.1	9.8 ± 1.3	10.5 ± 1.0
Insulin	1.2 ± 0.3	3.1 ± 0.4	1.8 ± 0.2	16.2 ± 2.1	7.9 ± 0.9	14.8 ± 1.7
PIG41	4.4 ± 0.5	6.9 ± 0.8	4.5 ± 0.6	15.3 ± 1.9	5.2 ± 0.6	16.7 ± 1.9
CBDP	3.3 ± 0.4	7.1 ± 0.8	3.8 ± 0.4	4.9 ± 0.6	1.6 ± 0.2	4.5 ± 0.6
Glim	3.1 ± 0.4	5.5 ± 0.7	2.9 ± 0.4	6.6 ± 0.8	2.2 ± 0.3	6.9 ± 1.0
Trypsin/NaCl	2.5 ± 0.4	6.3 ± 0.7	3.0 ± 0.4	4.2 ± 0.5	1.4 ± 0.3	4.5 ± 0.5
NEM	2.2 ± 0.4	5.0 ± 0.4	2.4 ± 0.3	5.2 ± 0.8	1.9 ± 0.3	4.6 ± 0.6

Isolated rat adipocytes were incubated (75 min, 37°C) in the absence (control) or presence of 1.5U/ml cholesterol oxidase (CO). Subsequently, portions of the CO-treated cells were washed by flotation and then incubated for the periods indicated. Thereafter, the adipocytes were incubated (15 min, 37°C) with 1 mM Na₃VO₄, 5 nM insulin, 3µM PIG41, or 10 µM glimepiride or electroporated in the presence of 300 µM caveolin-binding domain peptide (CBDP) or treated with trypsin plus NaCl or NEM. lcDIGs were prepared and immunoblotted for pp59^Lyn. IRS-1 was immunoprecipitated from lcDIGs and immunoblotted for tyrosine phosphorylation. Fold stimulations are given with basal values (absence of insulin) obtained with control cells set at 1. Each value represents the mean ± SD of two independent adipocyte preparations with incubations in triplicate each.

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