β-Arrestin 1 down-regulation after insulin treatment is associated with supersensitization of β2 adrenergic receptor Gαs signaling in 3T3-L1 adipocytes

Abstract

3T3-L1 adipocytes were incubated in serum-free media at 37°C with (dashed line) or without (solid line) 100 ng͞ml insulin for 8 h. Isoproterenol (10 M) was then added for the indicated time course at 37°C, and intracellular cAMP was determined by enzyme immunoassay, as described in Materials and Methods. Data are from a typical experiment done in triplicate wells Ϯ SEM and are representative of three separate experiments. (B) 3T3-L1 adipocytes were incubated in serum-free media for 8 h. Where indicated, 100 ng͞ml insulin was added for 15 min before isoproterenol treatment. Cells were then treated with 10 M isoproterenol for 5 min at 37°C, and intracellular cAMP was determined by enzyme immunoassay, as described in Materials and Methods. Data are from a typical experiment done in triplicate wells Ϯ SEM and are representative of three separate experiments.www.pnas.org͞cgi͞doi͞10.1073͞pnas.0630528100 After this, cells were treated with 10 M isoproterenol for 5 min at 37°C. Cells were then washed with PBS at 37°C and restimulated with a second 10 M dose of isoproterenol for an additional 5 min at 37°C; intracellular cAMP was determined by enzyme immunoassay. (B) 3T3-L1 adipocytes were incubated at 37°C in serum-free media for 8 h in the presence (dashed lines) or absence (solid lines) of 100 ng͞ml insulin. Forskolin was added for the final 5 min, and intracellular cAMP levels were determined by enzyme-linked immunoassay. Data are from typical experiments done in triplicate wells and are representative of three separate experiments.

β-Arrestin-1 Competitively Inhibits Insulin-Induced Ubiquitination and Degradation of Insulin Receptor Substrate 1

“…In keeping with this, we find that dephosphorylated ␤-arrestin is necessary for inhibition of IRS-1 degradation, since ␤-arrestin 412D, which constitutively mimics the phosphorylated state, does not cause this effect. This is also fully consistent with earlier results (4,12) showing that insulin stimulation leads to ␤-arrestin-1 Ser412 phosphorylation and degradation. This provides two coordinate mechanisms (inactivation [412 phosphorylation] and degradation) whereby chronic insulin treatment modulates the ␤-arrestin system to promote IRS-1 downregulation.…”

“…We have recently reported that long-term treatment of cells with insulin leads to ubiquitination and degradation of ␤-arrestin (4,12). To investigate whether the degradation of ␤-arrestin-1 could affect the process of insulin-mediated homologous desensitization, we examined the expression level of insulin signaling molecules, including the insulin receptor, IRS-1, IRS-2, Shc, and G␣q/11, with or without overexpression of wild-type (WT) ␤-arrestin-1 after insulin treatment for up to 16 h. As seen in Fig.…”

Antidiabetic effect of novel modulating peptides of G-protein-coupled kinase in experimental models of diabetes

“…It is known that the β2-adrenergic receptor is down-regulated by GRK2 and this receptor subtype has a significant role in regulating lipid metabolism in adipocytes [27,28,29]. In people with insulin resistance, there is a decrease in lipolysis due to a reduction in β2-adrenergic receptor in fat cells.…”

AKAP signaling complexes: getting to the heart of the matter

“…Insulin stimulation also activates PDE3B in a phosphatidylinositol-3-kinase (PI3-kinase)-and protein-kinase-B (PKB)-dependent manner, thereby reducing lipolysis [53,57,58]. Chronic insulin treatment of adipocytes, however, has been reported to increase isoproterenolinduced cAMP generation [59]. The effect of insulin treatment on cAMP-induced activation of PKA was measured using the PKA-activity reporter AKAR2 in 3T3-L1 adipocytes [4].…”