Impairment of PI3-K/Akt Pathway Underlies Attenuated Endothelial Function in Aorta of Type 2 Diabetic Mouse Model

Abstract

Abstract-The phosphatidylinositol 3-kinase (PI3-K) pathway, which activates serine/threonine protein kinase Akt, enhances endothelial nitric oxide synthase (eNOS) phosphorylation and nitric oxide (NO) production. We investigated the involvement of the PI3-K/Akt pathway in the relaxation responses to acetylcholine (ACh) and clonidine in a new type 2 diabetic model (streptozotocin plus nicotinamide-induced diabetic mice). Plasma glucose and insulin levels were significantly elevated in our model, and intravenous glucose tolerance tests revealed clear abnormalities in glucose tolerance and insulin responsiveness. Although in our model the ACh-induced relaxation and NO x Ϫ (NO 2 Ϫ ϩNO 3 Ϫ )/cGMP production were unchanged, the clonidine-induced and insulin-induced relaxations and NO x Ϫ /cGMP production were all greatly attenuated. In control mice, the clonidine-induced and insulin-induced relaxations were each abolished by LY294002 and by Wortmannin (inhibitors of PI3-K), and also by Akt-inhibitor treatment. The ACh-induced relaxation was unaffected by such treatments in either group of mice. The expression level of total Akt protein was significantly decreased in the diabetic mice aorta, but those for the p85 and p110␥ subunits of PI3-K were not. The clonidine-induced Ser-473 phosphorylation of Akt through PI3-K was significantly decreased in our model; however, that induced by ACh was not. These results suggest that relaxation responses and NO production mediated via the PI3-K/Akt pathway are decreased in this type 2 diabetic model. This may be a major cause of endothelial dysfunction (and the resulting hypertension) in type 2 diabetes. Key Words: diabetes mellitus Ⅲ hyperinsulinism Ⅲ aorta Ⅲ endothelium-derived relaxing factor Ⅲ nitric oxide N umerous epidemiological studies have indicated that the insulin resistance and hyperinsulinemia associated with type 2 diabetes make important contributions to the development of hypertension and cardiovascular diseases, and impaired endothelium-dependent vasodilation has been described in humans and in animal models of the disease. 1,2 We and others have demonstrated that both aortic endothelial dysfunction and hypertension are present in type 2 spontaneously diabetic (db/db Ϫ/Ϫ ) mice and in fructose-fed insulinresistance mice. [3][4][5][6] Our recent observation that endothelial function and nitric oxide (NO) production are impaired in aortic strips from spontaneously type 2 diabetic GotoKakizaki rats seemed to conflict with our finding that the expressions of the mRNA and protein for endothelial NO synthase (eNOS) were increased in such aortas. 7

Acetylcholine-induced vasodilation in the perfused kidney of the streptozotocin-induced diabetic rat: role of prostacyclin

“…decreased production of one of the EDRFs, enhanced inactivation of EDRF, impaired diffusion of EDRF to the underlying smooth muscle cells, decreased responsiveness of the smooth muscle to EDRF, and/or enhanced generation of EDCFs (Poston and Taylor, 1995;De Vriese et al, 2000b;Fitzgerald et al, 2005;Kobayashi et al, 2004Kobayashi et al, , 2005Matsumoto et al, 2004b). In vascular tissues in diabetes, ACh-induced relaxation appears to be blunted by the synthesis of an endothelium-derived constrictor prostanoid(s) (Tesfamariam et al, 1989).…”

“…In fact, renal hemodynamic changes may make an important contribution to diabetic nephropathy, and it is well known that vascular disease is one of the complicating features of diabetes mellitus in humans (Anderson and Vora, 1995;Sowers and Epstein, 1995). The reactivity of vascular smooth muscle and the endothelium to vasoactive agents has been extensively studied in diabetes in both experimental animals and humans (Poston and Taylor, 1995;De Vriese et al, 2000b;Fitzgerald et al, 2005;Kobayashi et al, 2004Kobayashi et al, , 2005Matsumoto et al, 2004b). Concerning the macrovasculature, an accumulating body of evidence suggests that the relaxation responses induced in the aorta by endothelium-dependent agents are weaker in streptozotocin (STZ)-induced diabetic animals than in non-diabetic control animals (Kamata et al, 1989De Vriese et al, 2000b;Kobayashi et al, 2000).…”

The PI3-K/Akt pathway: roles related to alterations in vasomotor responses in diabetic models

“…Several studies have suggested that the vasorelaxation induced by insulin or IGF-1 is most likely mediated by the production of vascular NO via the PI3-K/Akt pathway (Walsh et al, 1996;Kuboki et al, 2000;Kobayashi et al, 2004b). A recent study in our laboratory showed that addition of a PI3-K or Akt inhibitor did not significantly alter either ACh-induced relaxation or NOx/cyclic-GMP production in control aortae, whereas the clonidine-and insulin-induced responses were completely abolished by each inhibitor in such aortae (Kobayashi et al, 2004b). A study by Isenovic and coworkers (Isenovic et al, 2002) suggested that the wortomanninsensitive p85 regulatory subunit of PI3-K is involved in the isoproterenol-mediated relaxation of aortic rings via stimulation of eNOS activity.…”

“…However, a possible explanation may be that in the intact cells, NO synthesis is regulated independently of changes in eNOS enzyme activity. The most recent observations made in the our laboratory (Kobayashi et al, 2004b) compared aortae from nicotinamide-STZinduced type II diabetic mice with those from age-matched controls. Our findings were that (a) the diabetes altered neither ACh-induced relaxation nor ACh-induced NO/cyclic-GMP production (which are not mediated via the PI3-K/Akt signal pathways), while (b) the relaxations and NO/cyclic-GMP productions induced by clonidine or insulin were much weaker [passively, due to reductions in both Akt phosphorylation and Akt protein expression (but not to a decline in PI3-K activities)], in this type of diabetes.…”

Treatment of Spontaneously Hypertensive Rats With Rosiglitazone and/or Enalapril Restores Balance Between Vasodilator and Vasoconstrictor Actions of Insulin With Simultaneous Improvement in Hypertension and Insulin Resistance

“…Importantly, pathway-selective insulin resistance with impairment of insulin-stimulated PI 3-kinase signaling but not MAPK signaling is also present in vascular endothelium of insulin-resistant animal models (23)(24)(25). In spontaneously hypertensive rats (SHRs), a genetic model of hypertension with features of the metabolic syndrome, we recently demonstrated that metabolic insulin resistance is associated with impaired insulin-stimulated NO-dependent vasodilation (mediated by PI 3-kinase), as well as enhanced insulin-stimulated ET-1-dependent vasoconstriction (mediated by MAPK) (13).…”