Characterization of selective resistance to insulin signaling in the vasculature of obese Zucker (fa/fa) rats (original) (raw)
Ex vivo effect of insulin on tyrosine phosphorylation of IRs in the aorta and microvessels. Insulin’s effects on tyrosine phosphorylation of IRβ in isolated aorta and enriched microvessels (vascular stroma) isolated from Zucker lean (fa/+) and obese (fa/fa) rats were examined ex vivo. All the rats used in all the ex vivo experiments were 14 weeks old, and the obese rats were heavier than their age-matched lean control, with mean body weights of 482 ± 42 and 325 ± 20 g, respectively. The fasting blood glucose levels of the obese and lean rats were 108 ± 15 and 90 ± 12 mg/dL, respectively (Table 1).
The effect of insulin (2 or 100 nM) or control on tyrosine phosphorylation of IRβ was studied by immunoprecipitation with antibodies to IRβ and immunoblotted with αPY antibody. As shown in Figure 2, a and b, the basal levels of IRβ tyrosine phosphorylation were much lower in microvessels than in aorta from both lean and obese rats. Insulin, in a concentration-dependent manner, induced increases in IRβ tyrosine phosphorylation in microvessels from both lean and obese rats. In the lean rats, insulin at 2 and 100 nM increased IRβ tyrosine phosphorylation by 8.4- and 13-fold, respectively, compared with 3.2- and 5.6-fold increases in the microvessels from obese rats, representing reductions of 70% and 62% compared with lean rats. To determine whether the decreases in IRβ tyrosine phosphorylation in microvessels of obese rats were associated with decreases in the amount of the IRβ proteins, the same membranes were reblotted with antibodies to IRβ. As shown in Figure 2a (middle), protein levels of IR were not different between microvessels from Zucker lean and obese rats (Figure 2a).
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.
In isolated aorta immunoblot analysis of tyrosine-phosphorylated proteins in the αIRβ, immunoprecipitates showed that the basal tyrosine phosphorylation of IR was slightly higher in the aorta from obese rats than from lean rats (Figure 2b). Insulin-stimulated IRβ tyrosine phosphorylation was increased by 6.3-fold over the basal level in the aorta from lean rats, whereas only a 2.5-fold increase was observed in aorta from obese rats, a reduction of 71% compared with lean rats (P < 0.01). No significant differences were found in IR protein levels between the aorta of Zucker lean and obese rats (Figure 2b, middle).
Protein expression and tyrosine phosphorylation of IRS-1/2 and their association with p85 subunit of PI 3-kinase in the aorta. The protein levels of IRS-1 and IRS-2 in the aorta from control and obese rats were quantitated by immunoprecipitation and immunoblotting with αIRS-1 or αIRS-2 antibodies and showed a decrease of 23% and 34%, respectively, in the aorta of obese rats compared with lean controls (Figure 3a). In ex vivo experiments, insulin-stimulated (100 nM) increases of 3.6- and 5.3-fold in tyrosine phosphorylation of IRS-1 and IRS-2, respectively, in the aorta of lean rats (Figure 3, b and c), compared with increases of 1.9- and 2.5-fold, respectively, in the aorta of obese rats (P < 0.005; Figure 3, b and c), representing decreases of 65%. The same membranes used to detect tyrosine phosphorylation of IRS-1 and IRS-2 were reblotted with antibodies against p85 subunit of PI 3-kinase. In the lean rats increases of 3.7- and 4.5-fold in p85 association to IRS-1 and IRS-2, respectively, were induced by insulin (Figure 3, d and e), whereas only 1.6- and 1.9-fold increases were observed in the aorta of obese rats, reductions of 71–74% (P < 0.01).
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.
Ex vivo stimulation of PI 3-kinase activity in aorta and microvessels. Basal and insulin-stimulated PI 3-kinase activities were compared in IRS-1 or IRS-2 immunoprecipitates from the aorta and enriched microvessels of lean and obese rats in ex vivo experiments. Insulin (100 nM) increased IRS-1– and IRS-2–associated PI 3-kinase activities by 3.6- and 5.8-fold (n = 4) over the basal level in the aorta of lean rats (Figure 4, a and b), respectively, whereas insulin only stimulated increases of 1.8- and 2.1-fold in the aorta of obese rats. Thus, insulin-stimulated activities of PI 3-kinase associated with IRS-1 and IRS-2 were reduced by 69 and 77%, respectively, in the aorta of obese rats, compared with the lean rats. Insulin at 2 and 100 nM increased IRS-2–associated PI 3-kinase activities in enriched microvessels by 4- and 9-fold in lean rats and increased by only 1.5- and 3.5-fold in the obese rats, respectively (Figure 4c).
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.
Using immunoblotting analysis, no differences were noted between the total protein levels of the p85 subunit of PI 3-kinase in the aorta and microvessels from lean and obese rats. Similarly, no differences were detected between total PI 3-kinase activities in the aorta from lean and obese rats, as quantified by immunoprecipitation with antibodies to p85 subunit of PI 3-kinase (data not shown).
Insulin-stimulated serine phosphorylation of Akt/PKB in microvessels ex vivo. Akt is a serine/threonine kinase that is serine phosphorylated and can be activated by insulin through the PI 3-kinase pathway (34). In the microvessels from lean rats, insulin at 2 and 100 nM increased serine phosphorylation of Akt by 11- and 21-fold over the basal levels, respectively (Figure 5). However, in the microvessels from obese rats, insulin increased Akt serine phosphorylation by only 5- and 12-fold over the basal levels, respectively, reductions of 60% and 45% compared with lean rats.
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).
Physiological parameters of lean fa/+ and obese fa/fa Zucker rats used for euglycemic hyperinsulinemic clamp studies. Lean and obese rats were divided into insulin-treated and control groups, infused either with 10 mU/kg/min insulin or saline, respectively. During the study, blood glucose levels were monitored every 5 minutes and glucose was infused as needed to maintain blood glucose level at the preinfusion level. Fast insulin levels were 8 ± 2 and 262 ± 52 μU/mL in lean and obese rats, respectively, a difference of 30-fold. After infusion with insulin for 1 hour, serum insulin levels were significantly increased in both lean and obese rats (Table 1).
Effect of insulin on tyrosine phosphorylation of IRs in lean and obese rats in vivo. Euglycemic clamp study 1 (8 rats in each group) was performed to determine whether insulin resistance changed insulin-stimulated tyrosine phosphorylation of IRβ in aorta and liver. Figure 6a showed that insulin infusion increased IRβ tyrosine phosphorylation in the aorta of the lean rats by 4.2-fold, whereas increases of only 1.8-fold over the basal level were noted in the obese rats, a reduction of 75% (P < 0.01). Similarly, insulin also increased IRβ tyrosine phosphorylation by 7.5- and 3.0-fold in the livers of the lean and obese rats, respectively, a difference of 69% (P < 0.05) (Figure 6b). The relative abundance of IRβ subunits in the aorta and liver samples from lean and obese rats were also compared by using the same blots for the detection of IRβ tyrosine phosphorylation. As shown in Figure 6 (middle), IRβ protein levels in the aorta and liver were not different between lean and obese rats. Similarly, we did not find significant difference in IRβ protein levels in the skeletal muscle between lean and obese rats (data not shown).
(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).
The presence of IR/IGF-1 hybrid receptors was also detected in the microvessels. The abundance of hybrid receptors was significantly higher in obese than in lean rats (Figure 6c). However, the relative levels of the hybrid receptors in the microvessels accounted for only 10% and 16% of the total IR in lean and obese rats, respectively.
Effect of insulin on PI 3-kinase activities in lean and obese rats in vivo. Euglycemic clamp study 2 (5 rats in each group) was performed to determine the effect of insulin resistance on PI 3-kinase activation by insulin. As illustrated in Figure 7a, the mean levels of basal PI 3-kinase activities in the anti–IRS-2 immunoprecipitates were comparable in the aorta of lean and obese rats (P = 0.10; n = 5). Infusion with insulin (10 mU/kg/min for 1 hour) increased IRS-2–associated PI 3-kinase activities by 3.5-fold in the aorta of lean rats, compared with increases of only 1.7-fold in obese Zucker rats, a reduction of 72% (P < 0.01; n = 5 in each group).
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.
In the liver, the basal levels of PI 3-kinase activities of obese rats were significantly increased by 38%, compared with lean rats (P < 0.05; n = 5). Insulin increased IRS-2–associated PI 3-kinase activities (P < 0.01; n = 5) by 4.5- and 1.6-fold in the livers of lean and obese rats, respectively, a reduction of 82% (P < 0.01; n = 5 in each group) (Figure 7b).
Tyrosine phosphorylation of MAP kinase (ERK-1/2) in vascular tissues. Using antibodies against tyrosine-phosphorylated ERK-1/2, the levels of ERK-1/2 activation were examined in enriched microvessels after insulin stimulation in ex vivo experiments. As shown in Figure 8 (top), insulin (2 or 100 nM) stimulated tyrosine phosphorylation of ERK-1/2 equally in microvessels of both lean and obese rats. Insulin at 2 and 100 nM stimulated 3.0- and 4.3-fold increases above basal levels in tyrosine phosphorylation of ERK-1/2 in microvessels of lean rats, respectively (Figure 8, bottom), and 3.4- to 4.5-fold increases in the obese rats. The basal levels of tyrosine phosphorylation of ERK-1/2 were significantly higher in microvessels of obese rats than lean animals (P < 0.05; n = 6). No significant differences in ERK-1/2 protein levels in microvessels of obese and lean animals were observed (Figure 8, middle).
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.
ERK-1/2 tyrosine phosphorylations were examined in the aorta and liver after euglycemic hyperinsulinemic clamp. Insulin infusion did not stimulate tyrosine phosphorylation of ERK-1/2 in the aorta and liver from lean and obese rats. Interestingly, the basal levels of phosphorylated ERK-1/2 in the aorta and liver of obese rats were 2.2-fold (P < 0.05; n = 5) and 2.6-fold (P < 0.01; n = 5) higher, respectively, than those from lean controls (data not shown). The protein levels of ERK-1/2 were not significantly different in the aorta and liver between lean and obese animals.