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

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The underlying mechanism and the therapeutic regimen for the transition of reversible gingivitis to irreversible periodontitis are unclear. Since transforming growth factor (TGF)-β has been implicated in differentially regulated gene expression in gingival fibroblasts, we hypothesized that TGF-β signaling is activated in periodontitis-affected gingiva, along with enhanced collagen degradation, that is reversed by TGF-β inhibition. A novel three-dimensional (3D) gel-culture system consisting of primary human gingival fibroblasts (GF) and gingival epithelial (GE) cells in collagen gels was applied. GF populations from patients with severe periodontitis degraded collagen gels, which was reduced by TGF-β-receptor kinase inhibition. Up-regulation of TGF-β-responsive genes was evident in GF/GE co-cultures. Furthermore, the TGF-β downstream transducer Smad3C was highly phosphorylated in periodontitis-affected gingiva and 3D cultures. These results imply that TGF-β signaling is involved in fibroblast-epithelial cell interaction in periodontitis, and suggest that the 3D culture system is a useful in vitro model for therapeutic drug screening for periodontitis.

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Abstract. Type 2 diabetes mellitus is associated with high mortality and morbidity, mainly due to coronary artery disease and atherosclerosis, although female gender is a protective factor in the development of, for example, atherosclerosis and hypertension. Our main aim was to investigate gender differences in endothelial function in aortas from type 2 diabetic model mice. The nonfasting plasma glucose level was significantly elevated in diabetic mice (both males and females). The plasma insulin level was not different between controls and diabetics (either gender). The plasma adiponectin level was decreased by diabetes, and was lower in males. In control aortas (from males or females), the clonidine-induced relaxation was abolished by Aktinhibitor treatment. In diabetic males (versus both control males and diabetic females): a) the clonidine-and insulin-induced endothelium-dependent aortic relaxations were impaired, but the acetylcholine (ACh)-induced and sodium nitroprusside (SNP)-induced aortic relaxations were not, b) the norepinephrine (NE)-induced aortic contractile response was enhanced, c) systemic blood pressure was elevated, and d) the clonidine-stimulated Ser-473 phosphorylation of Akt in the aorta was decreased. These results suggest that endothelial functions dependent on the Akt pathway are abrogated by type 2 diabetes only in male mice.

Diabetes mellitus is a syndrome of disordered metabolism, usually due to a combination of hereditary and environmental causes, resulting in abnormally high blood glucose levels. The two most common forms of diabetes are due to either a diminished production of insulin (type 1) or a diminished response to insulin (type 2). Diabetes is a risk factor in the development of both macro-and microvascular diseases. [1][2][3] Indeed, it increases the incidence of ischemic heart disease, cerebral ischemia, and atherosclerosis, conditions in which endothelial dysfunction plays a role in pathogenesis. One of the most important functions of the endothelium is the production of nitric oxide (NO) in response to various hormonal, mechanical, and chemical stimuli. NO has a variety of effects, including the induction of vascular relaxation. In patients with either type 1 or type 2 diabetes, the forearm blood-flow (dilator) responses to acetylcholine (ACh) are reduced, suggesting endothelial dysfunction. 4,5) In streptozotocin (STZ)-induced diabetic rats or mice, which are models of type 1 diabetes, the endothelium-dependent relaxation induced by ACh is impaired, and reactive oxygen species and polyol products have been suggested to be involved in this impairment.6-9) Moreover, there is evidence that endothelial function is impaired in patients with diabetes mellitus 10,11) in a manner similar to that seen in STZ-treated animals. Thus, STZ-induced diabetic rats and mice may provide models for studies of the underlying causes of endothelial-cell damage in diabetes mellitus, and a means of assessing the contribution of diabetes mellitus to cardiovascular disease states.Gender differences in blood pressure 12) and vascular reactivity [13][14][15][16][17] have been reported in rats and mice. Although in the general (non-diabetic) population, cardiovascular diseases are more frequent in men than in premenopausal women, 18,19) diabetes may produce a greater impairment in the female cardiovascular system, with the result that the above difference between men and premenopausal women disappears in diabetic patients. 20) Although some studies have been carried out on the effects of exogenous sex hormones in diabetes, few studies have compared the influence of diabetes on vascular reactivity between males and females.Against this background, we decided to assess whether there are gender differences in the effects of type 1 diabetes on the endothelium-dependent vascular relaxations to various stimuli. For this, we employed aortas isolated from STZ-induced diabetic mice. Although there have been many reports that male diabetic-model mice or rats exhibit endothelial dysfunction, little is known about gender differences in endothelial function in diabetes, particularly in murine models. That was our rationale for investigating gender differences in vascular reactivity in the aorta using STZ-induced type 1 diabetic-model mice. MATERIALS AND METHODSReagents STZ, clonidine hydrochloride, insulin from porcine pancreas, norepinephrine (NE), and N w -...

Abstract. We investigated the effects of chronic simvastatin treatment on the impaired endothelium-dependent relaxation seen in aortas from type 2 diabetic mice. Starting at 8 weeks of diabetes, simvastatin (10 mg / kg per day) was administered to diabetic mice for 4 weeks. The significantly elevated systolic blood pressure in diabetic mice was normalized by simvastatin. Aortas from diabetic mice, but not those from simvastatin-treated diabetic mice, showed impaired endothelium-dependent relaxation in response to both clonidine and adrenomedullin. After preincubation with an Akt inhibitor, these relaxations were not significantly different among the three Akt inhibitor-treated groups (controls, diabetics, and simvastatin-treated diabetics). Although clonidine-induced NO x − (NO 2 − + NO 3 − ) production was greatly attenuated in our diabetic model, it was normalized by simvastatin treatment. The expression levels of both total Akt protein and clonidine-induced Ser-473-phosphorylated Akt were significantly decreased in diabetic aortas, while chronic simvastatin administration improved these decreased levels. The expression level of clonidine-induced phosphorylated PTEN (phosphatase and tensin homolog deleted on chromosome ten) was significantly increased in diabetic aortas, but chronic simvastatin did not affect it. These results strongly suggest that simvastatin improves the endothelial dysfunction seen in type 2 diabetic mice via increases in Akt and Akt phosphorylation.