In the present study we have characterized insulin signaling in the aorta and microvessels of lean and obese Zucker fa/fa
rats. The obese Zucker rats have insulin resistance, hyperinsulinemia, hyperlipidemia, glucose intolerance, and mild hypertension, properties similar to obesity-related insulin resistance and type 2 diabetes mellitus (35
). These results show that tyrosine phosphorylation of IR and IRS-1/2, PI 3-kinase activity, and serine phosphorylation of Akt/PKB are impaired in the vasculature of the obese animals. However, insulin stimulates tyrosine phosphorylation of ERK-1/2 MAP kinase equally in the microvessels of both lean and obese rats, although the basal levels of tyrosine phosphorylation of MAP kinase are significantly higher in the aorta and microvessels of the obese rats than in lean rats. Thus, we believe, these results have provided the first quantitative analysis of insulin-signaling pathways in intact vascular tissues, and have demonstrated a selective impairment of insulin’s activation on the PI 3-kinase-Akt pathway in the obese and insulin-resistant animals. In addition, both endothelial and smooth muscle cells are probably contributing to the parameters of the insulin signal measured in this study. We have previously reported that insulin can increase PI 3-kinase activation through IRS-1/2 in smooth muscle cells (30
); recently, we have found that endothelial cells have the same properties (data not shown).
The sites of insulin-signaling impairment appear to be at the IR and postreceptor levels in the aorta and microvessels of obese Zucker rats. Although the total amount of the receptors are not changed, the levels of IR tyrosine phosphorylation induced by insulin are decreased significantly in the blood vessels of obese Zucker rats (as examined by both in vivo and ex vivo studies) compared with lean rats. The reduction in IR tyrosine phosphorylation in the vascular cells is similar to that observed in liver, as examined by hyperinsulinemic euglycemic clamp study. Interestingly, the amount of IR/IGF-1R hybrid receptors may have been increased in the obese rats. These hybrid receptors may be less sensitive to insulin’s activation because they have lower affinity for insulin binding than homodimeric IR (36
). However, the contribution of the hybrid receptors to IR signaling in the vascular tissues may not be too significant, because the relative amounts of hybrid receptors to total IR were 1:10.
Possibly as a result of the decreased IR tyrosine phosphorylation, IRS-1 and IRS-2 tyrosine phosphorylation and PI 3-kinase activation in the aorta and microvessels of the obese Zucker rats are decreased. Decreases in protein levels of IRS-1 and IRS-2 by 23–34% in vascular tissues of the obese Zucker rats also contribute to the decline in PI 3-kinase activities. However, the total protein levels of PI 3-kinase and the total activities of PI 3-kinase immunoprecipitated with anti-p85 antibody are not altered in the vasculature of the obese rats, compared with lean rats. The decreases in IRS-1– and IRS-2–associated PI 3-kinase activities may be a consequence of changes in both IR kinase and decreased protein expression of IRS-1 and IRS-2.
Reductions in IR phosphorylation and signaling via PI 3-kinase pathway in the vascular tissues of obese Zucker rats appear to be similar to that found in muscle, liver, and adipose tissues of insulin-resistant animal models and humans (33
). In the liver of obese Zucker fa/fa
rats, insulin binding to the plasma membranes has been reported to be decreased with age in one study (42
), whereas other studies noted no changes in receptor binding, although receptor tyrosine kinase activity was decreased (43
). In obese patients IR numbers or binding are decreased in skeletal muscle (40
). However, insulin-stimulated PI 3-kinase activity is always impaired, and protein levels of IRS-1 and IRS-2 are decreased in muscle and liver of obese Zucker rats (38
). In diabetic ob/ob
mice, decreases in receptor tyrosine phosphorylation, IRS-1 tyrosine phosphorylation, and PI 3-kinase activation in liver and skeletal muscles are associated with the reduction in protein expression of IR, IRS-1 and IRS-2, and p85 subunit of PI 3-kinase (33
). In contrast, we did not find changes in protein expression of IRβ and p85 subunit of PI 3-kinase in vascular tissues of the obese Zucker rats. This difference could be because our results quantified total amount of IR rather than just that on the plasma membrane. In addition, it is possible that insulin resistance can be induced even without changes in IR protein level. For example, it has been reported that TNF-α inhibits IR signaling at IR and IRS-1 tyrosine phosphorylation without changing IR protein levels (45
There are probably several mechanisms that can cause decreases in insulin signaling via the PI 3-kinase pathway in the vasculature of obese insulin-resistant rats. First, increases in serine and threonine phosphorylation of IRβ may reduce receptor kinase activity and autophosphorylation. It has been reported that activation of PKC induces serine phosphorylation of IR, which can inhibit its tyrosine kinase activity, and leads to the decreases of insulin-induced PI 3-kinase activities both in vascular cells and nonvascular cells (46
). Activation of protein kinase C-α, -β, -ε, and -δ have been reported in skeletal muscle of Zucker obese and diabetic Goto-Kakizaki rats (48
). Second, altered phosphotyrosine phosphatase activity might lead to reduction in tyrosine phosphorylation of IR and IRS protein that secondarily decreases PI 3-kinase activity. Both increase and decrease in phosphotyrosine phosphatase activities have been reported in skeletal muscle and/or adipose tissue of Zucker obese rats and in obese and type 2 diabetic patients (49
). Third, dyslipidemia may play a role in mediating insulin resistance observed in Zucker obese rats. Recent studies suggested that experimentally induced hypertriglyceridemia and high plasma free fatty acid levels impair in vivo insulin’s metabolic action (52
). Last, circulating factors and hormone may also regulate insulin signaling in the vasculature. These cytokines include TNF-α and TNF-β, angiotensin II, and endothelin-1. TNF-α has been suggested as an important mediator of insulin resistance in obese animals through its overexpression from fat tissue (45
). Angiotensin II has been reported to inhibit insulin signaling through modulating IRS protein phosphorylation and/or PI 3-kinase activity (54
). Our recent study has also demonstrated that endothelin-1, which is elevated in type 2 diabetes, attenuated insulin-stimulated PI 3-kinase activity in cultured VMSCs (30
Selective impairment of insulin signaling on PI 3-kinase pathway in the vascular tissues could be pathophysiologically important in the development of cardiovascular diseases. A recent study by Zeng and Quon showed that activation of PI 3-kinase pathway could be involved in insulin’s stimulatory effect on NO release in cultured vascular endothelial cells (3
). This effect of insulin on NO could be responsible for insulin’s vasodilatory actions (7
). In an insulin-resistant state, the vasodilatation induced by insulin is blunted because the PI 3-kinase pathway is partially inhibited. Interestingly, the vasodilatory effect of insulin is also blunted in subjects with obesity and patients with type 2 diabetes (12
). In addition, we have recently reported that insulin’s vasodilatory effect could be partially due to increases in eNOS gene expression via PI 3-kinase pathway (6
). Besides vasodilatory actions, stimulation of NO production by insulin may also affect vascular remodeling, such as inhibiting VSMC proliferation and migration and inhibiting platelet aggregation and leukocyte adhesion to endothelial cells (55
). These reports suggest that the inhibition of PI 3-kinase pathway activation by insulin in vascular tissues of insulin-resistant states may contribute to the loss of insulin’s effect on NO production, which will enhance the atherogenic potential of the insulin-resistant state.
In contrast to PI 3-kinase activation, much less is known about insulin’s activation of ERK-1/2 MAP kinase in insulin-resistant or diabetic conditions in vivo. Our results have established that insulin at physiological levels can activate MAP kinase in microvessels, although to a modest level. Surprisingly, the effect of insulin on ERK-1/2 MAP kinase tyrosine phosphorylation is not observed in the aorta and liver in vivo by using the euglycemic clamp. This could be the result of the lack of an effective way to isolate the MAP kinase pool specifically activated by insulin in euglycemic clamp studies. Alternatively, manipulation of the tissues during isolation could have artificially activated MAP kinase and mimicked insulin’s effect. Because insulin’s effect on MAP kinase is relatively weak compared with other growth factors such as PDGF, it is possible that insulin growth effect mediated by MAP kinase activation may not be pathophysiologically important. This is consistent with insulin being a very weak stimuli of vascular cell proliferation at physiological concentrations, compared with other growth factors (56
We believe that these studies have provided the first direct evidence that insulin can activate both the PI 3-kinase and MAP kinase pathways in the vasculature ex vivo or in vivo. In an insulin-resistant state, PI 3-kinase pathway appears to be selectively blunted in vascular tissues, compared with MAP kinase activity. These findings have provided support for the hypothesis that under physiological states, insulin may have vasodilatory and antiatherogenic actions mediated by the PI 3-kinase–NO pathway, although activation of PI 3-kinase may also stimulate DNA synthesis (20
). In the obese insulin-resistant state, insulin’s vasodilatory and antiatherogenic functions, mediated through PI 3-kinase pathway, may be impaired, whereas proatherogenic actions mediated through MAP kinase cascade may be increased due to hyperinsulinemia and plasma mitogenic factors. The imbalance between PI 3-kinase and MAP kinase signal pathways in the vasculature may lead to the development of cardiovascular abnormality observed in insulin resistance and diabetic patients.