In obese patients with type 2 diabetes, insulin delivery to and insulin-dependent glucose uptake by skeletal muscle are delayed and impaired. The mechanisms underlying the delay and impairment are unclear. We demonstrate that impaired insulin signaling in endothelial cells, due to reduced Irs2 expression and insulin-induced eNOS phosphorylation, causes attenuation of insulin-induced capillary recruitment and insulin delivery, which in turn reduces glucose uptake by skeletal muscle. Moreover, restoration of insulin-induced eNOS phosphorylation in endothelial cells completely reverses the reduction in capillary recruitment and insulin delivery in tissue-specific knockout mice lacking Irs2 in endothelial cells and fed a high-fat diet. As a result, glucose uptake by skeletal muscle is restored in these mice. Taken together, our results show that insulin signaling in endothelial cells plays a pivotal role in the regulation of glucose uptake by skeletal muscle. Furthermore, improving endothelial insulin signaling may serve as a therapeutic strategy for ameliorating skeletal muscle insulin resistance.

Nitric oxide (NO) is produced in almost all tissues and organs, exerting a variety of biological actions under physiological and pathological conditions. NO is synthesized by three different isoforms of NO synthase (NOS), including neuronal, inducible, and endothelial NOSs. Because there are substantial compensatory interactions among the NOS isoforms, the ultimate roles of endogenous NO in our body still remain to be fully elucidated. Here, we have successfully developed mice in which all three NOS genes are completely deleted by crossbreeding singly NOS ؊/؊ mice. NOS expression and activities were totally absent in the triply NOS ؊/؊ mice before and after treatment with lipopolysaccharide. Although the triply NOS ؊/؊ mice were viable and appeared normal, their survival and fertility rates were markedly reduced as compared with the wild-type mice. Furthermore, these mice exhibited marked hypotonic polyuria, polydipsia, and renal unresponsiveness to an antidiuretic hormone, vasopressin, all of which are characteristics consistent with nephrogenic diabetes insipidus. In the kidney of the triply NOS ؊/؊ mice, vasopressin-induced cAMP production and membranous aquaporin-2 water channel expression were reduced associated with tubuloglomerular lesion formation. These results provide evidence that the NOS system plays a critical role in maintaining homeostasis, especially in the kidney.

Objective-Asymmetric dimethylarginine (ADMA) is widely believed to be an endogenous nitric oxide synthase (eNOS) inhibitor. However, in this study, we examined our hypothesis that the long-term vascular effects of ADMA are not mediated by inhibition of endothelial NO synthesis. Methods and Results-ADMA was infused in wild-type and eNOS-knockout (KO) mice by osmotic minipump for 4 weeks. In wild-type mice, long-term treatment with ADMA caused significant coronary microvascular lesions. Importantly, in eNOS-KO mice, treatment with ADMA also caused an extent of coronary microvascular lesions that was comparable to that in wild-type mice. These vascular effects of ADMA were not prevented by supplementation of L-arginine, and vascular NO production was not reduced by ADMA treatment. Treatment with ADMA caused upregulation of angiotensin-converting enzyme (ACE) and an increase in superoxide production that were comparable in both strains and that were abolished by simultaneous treatment with temocapril (ACE inhibitor) or olmesartan (AT 1 receptor antagonist), which simultaneously suppressed vascular lesion formation. Key Words: asymmetric dimethylarginine Ⅲ arteriosclerosis Ⅲ nitric oxide Ⅲ endothelial nitric oxide synthase Ⅲ mice E ndothelium-derived nitric oxide (NO), synthesized from L-arginine by endothelial NO synthase (eNOS), has several important antiatherogenic actions. 1-5 Indeed, reduction of endothelial NO synthesis (endothelial dysfunction) predisposes the blood vessel to arteriosclerosis, 1-5 and the eNOS-deficient (eNOS-KO) mice exhibit accelerated vascular lesion formation. 6,7 As pharmacological tools to inhibit endothelial NO synthesis, synthetic L-arginine analogues have been used in vitro and in vivo. Among them, N -nitro-L-arginine methyl ester (L-NAME) is the most frequently used agent. [1][2][3][4][5] Long-term treatment with L-NAME is known to cause arteriosclerotic coronary lesions, especially at microvascular levels, in experimental animals. 8,9 This model with L-NAME is regarded as a useful animal model for examining the protective roles of endothelium-derived NO in the pathogenesis of arteriosclerosis. 8,9 See cover However, it is controversial whether these vascular effects of L-NAME are caused primarily by the inhibition of endothelial NO synthesis for the following reasons: first, the importance of endothelium-derived NO decreases as the vessel size becomes smaller, 10 whereas L-NAME-induced vascular lesions are prominent at microvascular levels; 8 second, long-term treatment with L-NAME does not reduce eNOS activity; 11 third, multiple actions of L-NAME other than simple inhibition of NO synthesis have been reported. 12,13 The most appropriate way to address this issue is to use mice that are deficient in the eNOS gene and to examine whether long-term treatment with L-NAME causes coronary vascular lesions in those mice. We have recently shown that treatment with L-NAME causes a comparable extent of Conclusions-These

The endothelium plays an important role in maintaining vascular homeostasis by synthesizing and releasing several relaxing factors, such as prostacyclin, nitric oxide (NO), and endothelium-derived hyperpolarizing factor (EDHF). We have previously demonstrated in animals and humans that endothelium-derived hydrogen peroxide (H 2 O 2 ) is an EDHF that is produced in part by endothelial NO synthase ( eNOS). In this study, we show that genetic disruption of all three NOS isoforms (neuronal [nNOS], inducible [iNOS], and endothelial [eNOS]) abolishes EDHF responses in mice.The contribution of the NOS system to EDHFmediated responses was examined in eNOS ؊ / ؊ , n/eNOS ؊ / ؊ , and n/i/eNOS ؊ / ؊ mice. EDHFmediated relaxation and hyperpolarization in response to acetylcholine of mesenteric arteries were progressively reduced as the number of disrupted NOS genes increased, whereas vascular smooth muscle function was preserved. Loss of eNOS expression alone was compensated for by other NOS genes, and endothelial cell production of H 2 O 2 and EDHF-mediated responses were completely absent in n/i/eNOS ؊ / ؊ mice, even after antihypertensive treatment with hydralazine. NOS uncoupling was not involved, as modulation of tetrahydrobiopterin (BH 4 ) synthesis had no effect on EDHF-mediated relaxation, and the BH 4 /dihydrobiopterin (BH 2 ) ratio was comparable in mesenteric arteries and the aorta. These results provide the fi rst evidence that EDHF-mediated responses are dependent on the NOSs system in mouse mesenteric arteries.