Characterization of selective resistance to insulin signaling in the vasculature of obese Zucker (fa/fa) rats

Abstract

Both insulin resistance and hyperinsulinemia have been reported to be independent risk factors for cardiovascular diseases. However, little is known regarding insulin signaling in the vascular tissues in insulin-resistant states. In this report, insulin signaling on the phosphatidylinositol 3-kinase (PI 3-kinase) and mitogen-activated protein (MAP) kinase pathways were compared in vascular tissues of lean and obese Zucker (fa/fa) rats in both ex vivo and in vivo studies. Ex vivo, insulin-stimulated tyrosine phosphorylation of insulin receptor beta subunits (IRbeta) in the aorta and microvessels of obese rats was significantly decreased compared with lean rats, although the protein levels of IRbeta in the 2 groups were not different. Insulin-induced tyrosine phosphorylation of insulin receptor substrates 1 and 2 (IRS-1 and IRS-2) and their protein levels were decreased in the aorta of obese rats compared with lean rats. The association of p85 subunit to the IRS proteins and the IRS-associated PI 3-kinase activities stimulated by insulin in the aorta of obese rats were significantly decreased compared with the lean rats. In addition, insulin-stimulated serine phosphorylation of Akt, a downstream kinase of PI 3-kinase pathway, was also reduced significantly in isolated microvessels from obese rats compared with the lean rats. In euglycemic clamp studies, insulin infusion greatly increased tyrosine phosphorylation of IRbeta- and IRS-2-associated PI 3-kinase activity in the aorta of lean rats, but only slight increases were observed in obese rats. In contrast, insulin stimulated tyrosine phosphorylation of MAP kinase (ERK-1/2) equally in isolated microvessels of lean and obese rats, although basal tyrosine phosphorylation of ERK-1/2 was higher in the obese rats. To our knowledge, these data provided the first direct measurements of insulin signaling in the vascular tissues, and documented a selective resistance to PI 3-kinase (but not to MAP kinase pathway) in the vascular tissues of obese Zucker rats.

Figure 1

Microvessels isolated from rat epididymal fat-pads. Isolated epididymal fat-pads were digested with collagenase and homogenized as described in Methods. The purity of the isolated microvessels were verified by histochemical staining with H&E (a) and immunohistochemical staining with factor VIII antibody (b). Immunoblotting with antibody against vascular α-SMA was used to compare the quality of isolated microvessels from lean and obese rats (c).

Figure 2

Tyrosine phosphorylation and protein levels of IRβ in the aorta and microvessels of obese Zucker fa/fa and lean rats ex vivo. Microvessels (a) and aorta (b) were isolated from lean and obese rats as described in Methods, followed by incubation with or without insulin (2–100 nM) in DMEM (0.1% BSA) for 5 minutes (microvessels) or 30 minutes (aorta) at 37°C. Equal amounts of protein (6 mg of microvessel sample and 4 mg of aorta sample) were subjected to immunoprecipitation with αIRβ antibody, separated by SDS-PAGE, and immunoblotted with αPY antibody (top panels). Stripped membranes were reblotted with αIRβ antibody (middle panels). Data (mean ± SD; n = 4) are expressed as relative to control, assigning a value of 100% to the lean control mean. *P < 0.05, lean vs. obese tissues incubated with insulin at the same concentrations.

Figure 3

Protein levels, tyrosine phosphorylation of IRS-1 and IRS-2, and their association with P85 subunit of PI 3-kinase in the aorta ex vivo. (a) Protein levels of IRS-1 and IRS-2. Isolated aortas were homogenized, and equal amounts of protein (10 mg) were subjected to immunoprecipitation with αIRS-1 or αIRS-2 antibodies, separated by SDS-PAGE, and immunoblotted with the same antibody. Data (mean ± SD; n = 4) are expressed as relative to control, assigning a value of 100% to the lean control mean. *P < 0.05, lean vs. obese. (b and c) Insulin-stimulated tyrosine phosphorylation of IRS-1 and IRS-2. Isolated aortas were incubated without or with insulin (100 nM) in DMEM (0.1% BSA) for 30 minutes at 37°C. Equal amounts of protein (6 mg) were subjected to immunoprecipitation with αIRS-1 or αIRS-2 antibodies, separated by SDS-PAGE, and immunoblotted with αPY antibodies. (d and e) Association of p85 subunit of PI 3-kinase to IRS-1 and IRS-2. The same membranes used for the IRS tyrosine phosphorylation study were stripped and reblotted with αp85 subunit antibody (top panels). Data (mean ± SD; n = 4) in b–e are expressed as relative to control, assigning a value of 100% to the lean insulin mean. *P < 0.05, **P < 0.01, insulin lean vs. insulin obese.

Figure 4

IRS-1– and IRS-2–associated PI 3-kinase activities in the aorta and microvessels ex vivo. Isolated aortas were incubated without or with insulin (100 nM) in DMEM (0.1% BSA) for 30 minutes at 37°C. Equal amounts of protein (2 mg) samples were subjected to immunoprecipitation with αIRS-1 (a) or αIRS-2 (b) antibodies. In the studies with microvessels (c), samples were treated without or with insulin (2–100 nM) in DMEM (0.1% BSA) for 5 minutes at 37°C, and 2-mg protein samples were subjected to immunoprecipitation with αIRS-2 antibody. The kinase activities were measured in the presence of phosphatidylinositol and [³²P]ATP, and the lipid products were separated by TLC (top panels). The spots that comigrated with a PI 3-phosphate (PIP) standard were quantified by a PhosphorImager. Data (mean ± SD; n = 4) are expressed as relative to control, assigning a value of 100% to the mean of 100-nM insulin-treated samples of lean rats. *P < 0.05, **P < 0.01, lean vs. obese treated with insulin at the same concentrations.

Figure 5

Insulin-stimulated serine phosphorylation of Akt in isolated microvessels ex vivo. Isolated microvessels were stimulated without or with insulin (2 and 100 nM) for 20 minutes at 37°C (n = 3; each sample of microvessels was derived from 2 rats). Tissues were homogenized in lysis buffer as described in Methods. Lysates (100 μg protein) were separated with 8% SDS-PAGE and transferred to nitrocellulose membrane. The membranes were blotted with anti-phospho-(serine 473)-Akt antibody, viewed using an ECL kit, and quantified with a densitometer. *P < 0.05, **P < 0.01, lean vs. obese treated with insulin at the same concentrations (n = 3; each sample of microvessels was derived from 2 rats).

Figure 6

(a and b) Tyrosine phosphorylation and protein levels of IRβ in the aorta and liver after euglycemic hyperinsulinemic clamp. Animals were infused with saline (control) or insulin (10 mU/kg/min) for 1 hour during a euglycemic clamp as described in Methods. The aorta and liver were removed, and equal amounts of protein were subjected to immunoprecipitation with IRβ antibody, separated by SDS-PAGE, and immunoblotted with αPY antibody (top panels). Stripped membranes were reblotted with αIRβ antibody (middle panels). Data (mean ± SD; n = 4) are expressed as relative to control, assigning a value of 100% to the lean control mean. *P < 0.05, insulin lean vs. insulin obese. (c) Hybrid IR/IGF-1R in microvessels of lean and obese rats. Isolated microvessels were homogenized, and lysates (6 mg protein) were subjected to immunoprecipitation with αIRβ or IGF-1Rβ antibodies, separated by SDS-PAGE, and immunoblotted with αIGF-1Rβ or αIRβ antibodies as indicated. Stripped membranes were reblotted with αIRβ antibody (middle). The percentage of hybrid receptors in total IR was calculated by the ratio between PhosphorImager counting of IGF-1Rβ bands (top) and IRβ bands (middle) in αIRβ immunoprecipitates. *P < 0.05, lean vs. obese rats (n = 3; each sample of microvessels was derived from 2 rats).

Figure 7

IRS-2–associated PI 3-kinase activities in the aorta and liver after euglycemic hyperinsulinemic clamp. Animals were infused with saline (control) or insulin (10 mU/kg/min) for 1 hour during a euglycemic clamp as described in Methods. IRS-2 associated PI 3-kinase in the aorta, and liver was immunoprecipitated with αIRS-2 antibody. The kinase activities were measured in the presence of phosphatidylinositol and [³²P]ATP, and the lipid products were separated by TLC (top panels). The spots that comigrated with PIP standard were quantified by a PhosphorImager. Data (mean ± SD; n = 5) are expressed relative to control, assigning a value of 100% to the control mean. *P < 0.05, insulin lean vs. insulin obese.

Figure 8

Effects of insulin on tyrosine phosphorylation of MAP kinase (ERK-1/2) in microvessels ex vivo. Isolated microvessels were incubated without or with insulin (2 or 100 nM) in DMEM (0.1% BSA) for 10 minutes at 37°C. Fifty micrograms of protein of each sample was separated by SDS-PAGE and immunoblotted with antibody specific for tyrosine-phosphorylated ERK-1/2. Stripped membranes were reblotted with polyclonal antibody against ERK-1/2 proteins. Data (mean ± SD; n = 6) are expressed relative to control, assigning a value of 100% to lean control mean.