Insulin receptor substrate (IRS) 1 is reduced and IRS-2 is the main docking protein for phosphatidylinositol 3-kinase in adipocytes from subjects with non-insulin-dependent diabetes mellitus

Abstract

The large docking protein IRS-1 is a major substrate for the insulin receptor and other tyrosine kinases. It plays a key role in eliciting many of insulin's actions, including binding and activation of phosphatidylinositol (PI) 3-kinase and the subsequent increase in glucose transport. Gene disruption of IRS-1 in mice is associated with an impaired insulin-stimulated glucose disposal in vivo and glucose transport in vitro, but the survival of the animals and residual insulin sensitivity is dependent on the presence of the alternative docking protein IRS-2. We examined the expression and function of IRS-1 and IRS-2 in adipocytes from healthy and diabetic individuals. Cells from subjects with non-insulin-dependent diabetes mellitus (NIDDM), but not with insulin-dependent diabetes mellitus, had an impaired insulin effect and a marked reduction (70 +/- 6%) in the expression of IRS-1 protein, whereas IRS-2 was unchanged. In normal cells, IRS-1 was the main docking protein for the binding and activation of insulin-stimulated PI 3-kinase; IRS-2 was also functional but required a higher insulin concentration for a similar binding and activation of PI 3-kinase. In contrast in NIDDM cells with a low IRS-1 content, IRS-2 became the main docking protein. These findings may provide important reasons for the insulin resistance in NIDDM.

Figure 1

Effect of various concentrations of insulin on glucose transport in adipocytes from healthy subjects and NIDDM subjects. Values represent the mean ± SEM of six experiments with triplicate determinations. The value at 100% is the basal uptake in the absence of insulin.

Figure 2

IRS-1 protein content in adipocytes from healthy subjects and IDDM and NIDDM subjects. (A and B) Cell lysates from adipocytes of healthy control, IDDM, or NIDDM subjects (50 μg of protein) were separated by SDS/PAGE (10% polyacrylamide gels) and immunoblotted with the indicated antibodies. Each lane represents the pooled lysates of two subjects, and each blot is representative of at least three experiments. (C) Cell lysates from adipocytes preincubated in the presence or absence of insulin (INS) were immunoprecipitated with antibodies against IRS-1 (C-terminal), separated by SDS/PAGE on 7.5% gels, and immunoblotted with the indicated antibodies.

Figure 3

Expression and function of IRS-2 in adipocytes from healthy control and NIDDM subjects. (A) Adipocytes from control or NIDDM subjects were incubated without or with 6.9 nM insulin (INS) for 10 min. Cell lysates were loaded by equal amount of proteins, separated by SDS/PAGE (10% gels), and immunoblotted with anti-phosphotyrosine (PY) antibodies. (B) Cell lysates were immunoprecipitated with anti-IRS-2 antibodies and immunoblotted with anti-IRS-2, anti-PY, anti-p85, and anti-Grb2 as indicated. (C) Supernatants from anti-IRS-2 immunoprecipitates were separated by SDS/PAGE (7.5% gels) and immunoblotted with anti-IRS-1 C-terminal antibodies. (D) PI 3-kinase activity was assayed on immunoprecipitates (IP) produced by the indicated antibodies on lysates from untreated or insulin-stimulated adipocytes from control or NIDDM subjects.

Figure 4

IRS-1 is the main docking protein for PI 3-kinase in human adipocytes. Adipocytes from healthy control subjects were incubated for 10 min with different concentrations of insulin. Cell lysates were immunoprecipitated with anti-IRS-1 C-terminal antibodies (A) or anti-IRS-2 antibodies (B) separated on 7.5% SDS-PAGE and immunoblotted with the indicated antibodies. (C) PI 3-kinase activity was assayed on immunoprecipitates produced by the indicated antibodies as described in Fig. 3. (D) Quantification of the PI 3-kinase activity using a PhosphorImager (Molecular Dynamics).