Enhancing eNOS activity with simultaneous inhibition of IKKβ restores vascular function in Ins2Akita+/− type-1 diabetic mice

Abstract

The balance of nitric oxide (NO) versus superoxide generation has a major role in the initiation and progression of endothelial dysfunction. Under conditions of high glucose, endothelial nitric oxide synthase (eNOS) functions as a chief source of superoxide rather than NO. In order to improve NO bioavailability within the vessel wall in type-1 diabetes, we investigated treatment strategies that improve eNOS phosphorylation and NO-dependent vasorelaxation. We evaluated methods to increase the eNOS activity by (1) feeding Ins2Akita spontaneously diabetic (type-1) mice with L-arginine in the presence of sepiapterin, a precursor of tetrahydrobiopterin; (2) preventing eNOS/NO deregulation by the inclusion of inhibitor kappa B kinase beta (IKKβ) inhibitor, salsalate, in the diet regimen in combination with L-arginine and sepiapterin; and (3) independently increasing eNOS expression to improve eNOS activity and associated NO production through generating Ins2Akita diabetic mice that overexpress human eNOS predominantly in vascular endothelial cells. Our results clearly demonstrated that diet supplementation with L-arginine, sepiapterin along with salsalate improved phosphorylation of eNOS and enhanced vasorelaxation of thoracic/abdominal aorta in type-1 diabetic mice. More interestingly, despite the overexpression of eNOS, the in-house generated transgenic eNOS-GFP (TgeNOS-GFP)-Ins2 Akita cross mice showed an unanticipated effect of reduced eNOS phosphorylation and enhanced superoxide production. Our results demonstrate that enhancement of endogenous eNOS activity by nutritional modulation is more beneficial than increasing the endogenous expression of eNOS by gene therapy modalities. Endothelial nitric oxide synthase (eNOS) that utilizes L-arginine as a substrate to generate nitric oxide (NO) is a key regulator of vascular function. Even though endothelial cells, a predominant source of eNOS exhibit constitutive enzyme activity, several other regulators are necessary to induce eNOS activity based on local demand for NO. Reduced bioavailability of NO that causes endothelial dysfunction is a hallmark of diabetes. Several mechanisms have been implicated causing 'endothelial dysfunction' in diabetes. The reduced availability of the substrate L-arginine and the cofactor tetrahydrobiopterin (BH4) has been implicated due to increased arginase activity and enhanced superoxide generation. 1 Uncoupling of eNOS has been shown to be central to robust superoxide generation and the associated vascular dysfunction. 2 Increased levels of asymmetric NG, NG-dimethyl arginine (ADMA-a substrate analog of L-arginine) in circulation have also been shown in diabetic patients limiting the availability of L-arginine to eNOS. 3 In vitro studies from our laboratory and reports from other investigators have shown that the regulation of eNOS activity is mediated by heat shock protein-90 (Hsp-90). 4,5 The interaction of eNOS with Hsp-90 favors the phosphorylation of eNOS at Ser1177 by Akt kinase. This phosphorylation process is a key event which a...

Effect of Treatment with Salsalate, Menhaden Oil, Combination of Salsalate and Menhaden Oil, or Resolvin D1 of C57Bl/6J Type 1 Diabetic Mouse on Neuropathic Endpoints

“…Furthermore, studies have shown salsalate treatment blocks free fatty acid mediated vascular insulin resistance [ 33 ] with improved glycemic control and with reduced inflammatory outcomes measures [ 17 , 20 ]. Some beneficial metabolic effects may be mediated by increased energy expenditure from activated brown adipose tissue [ 34 ] and reduced microvascular complications via inhibition of inhibitor of kappa B kinase β (IKK β ), the subsequent reduction in NF- κ B related genes, and increased nitric oxide (NO) preventing ischemic nerve damage [ 35 , 36 ]. This study demonstrated for the first time that salsalate alone can improve diabetic neuropathic endpoints in a mouse model of type 1 diabetes even when treatment was delayed for eight weeks after the onset of hyperglycemia.…”

Targeting Nitric Oxide with Natural Derived Compounds as a Therapeutic Strategy in Vascular Diseases

“…The eNOS posttranslational modifications are necessary also in mediating the antidiabetic effects of several therapeutic interventions. For example, a diet supplementation with l-arginine and sepiapterin along with salsalate has been proved to increase the eNOS phosphorylation and improved vasorelaxation of thoracic/abdominal aorta in type-1 diabetic mice [ 76 ]. Furthermore, Ding et al showed that cardiac overexpression of SIRT1, a NAD+-dependent deacetylases, reduced diabetes-exacerbated myocardial ischemia reperfusion injury and oxidative stress in diabetic rats via eNOS activation and that such effect was mediated by increase of the eNOS phosphorylation and reduction of the eNOS acetylation [ 77 ].…”

Targeted overexpression of endothelial nitric oxide synthase in endothelial cells improves cerebrovascular reactivity in Ins2Akita-type-1 diabetic mice

“…We studied only 6-month old animals, and future studies will investigate these animals at different age groups. There are also alternative approaches to increase eNOS activity, such as by diet supplementation of L-arginine, sepiapterin, and salsalate, 40 warranting further investigation. Future studies could include investigating neurovascular coupling associated with evoked functional stimulation.…”