Dietary Nitrate to Augment Exercise Training Benefits in DM+PAD

描述(申请人提供)：外周动脉疾病(PAD)影响5%的美国50岁以上的人口，其中三分之一患有间歇性跛行(IC)。糖尿病患者(DM+PAD)的PAD发生率大大增加，因此建议对无症状的糖尿病患者进行PAD筛查。糖尿病合并PAD患者的疾病进展为严重的腿部缺血、下肢截肢和心血管事件的风险增加。生物可利用的血管NO减少可能是糖尿病的一个特别相关的问题，因为已有证据表明，高血糖会减少内皮源性NO的产生，而晚期糖基化终产物和活性氧物种会使NO失活。我们之前已经证明，只有PAD，但不是2型糖尿病(T2D)+PAD，受试者能够在运动训练后上调血管NO的生物利用度和相关的血浆亚硝酸盐信号，这些变化介导了更明显的功能反应。我们还显示，与安慰剂相比，单剂量摄入高硝酸盐饮料后，仅服用PAD的COT和PWT分别增加了18%和17%，这会提高血浆亚硝酸盐水平。此外，在慢性治疗后：A)在小鼠模型中，血管生成和动脉生成的后肢缺血仅在缺血肢体显著增加；以及B)在健康人类治疗两天后，线粒体氧效率提高，从而降低了运动中的氧气成本。这项建议的假设是，在患有IC的T2D+PAD患者中，定期服用可升高血浆亚硝酸盐的高硝酸盐补充剂，结合12周的肢体缺血阈值(SET)的有监督的运动训练，将产生更大的临床益处(增加 COT和PWT)与接受相同运动训练但补充安慰剂(PET)的组进行比较。为了充分开发力量并进行更大规模的研究，以下特定目标将探索我们的假设，使用24名患有T2D+PAD和IC的个体(每组12人)：特定目标1：在12周的监督运动训练后，确定在最大分级运动试验中COT、PWT和VO2峰值变化的组间差异。具体目的2：确定在最大负荷运动试验中(A)组织氧合(NIRS)和(B)NO衍生物种(血浆和红细胞亚硝酸盐、硝酸盐、亚硫醇、血浆硝基酪氨酸)、cGMP和(C)静息血管功能(内皮功能)、小腿血流量(体积描记)变化的组间差异。具体目的3a：确定腓肠肌血管生成(单位面积毛细血管和每肌纤维毛细血管、内皮细胞与周围周细胞、I型与II型纤维的相对比例)变化的组间差异。如果存在差异，我们将寻找以下方面的变化：(A)细胞增殖(PCNA)和细胞凋亡(TUNEL)；(B)氧化能力(柠檬酸合成酶活性)；(C)线粒体的体积和密度；(D)线粒体的氧效率(呼吸控制率、ATP磷酸化率与耗氧率的比率(P/O比)和最大ATP产生速率)。

DESCRIPTION (provided by applicant): Peripheral artery disease (PAD) affects 5% of the US population over 50 yrs, one third of which suffer from intermittent claudication (IC). The incidence of PAD in subjects with diabetes (DM+PAD) is so greatly increased that screening for PAD in asymptomatic diabetic subjects is recommended. DM+PAD patients are at increased risk for disease progression to critical leg ischemia, lower extremity amputation, and cardiovascular events. Reduced bioavailable vascular NO may be a particularly pertinent issue in diabetes given that endothelial derived NO production has been shown to be decreased by hyperglycemia, and NO is inactivated by advanced glycation end products and reactive oxygen species. We have previously shown PAD-only, but not type 2 diabetic (T2D)+PAD, subjects are able to up-regulate vascular NO bioavailability and related plasma nitrite signaling following exercise training and that these changes mediate a more pronounced functional response. We have also shown an 18% and 17% increase in COT and PWT respectively in PAD-only, following a single dose ingestion of a high-nitrate containing beverage which raises plasma nitrite in comparison to placebo. Additionally, following chronic treatment; A) In a murine model hind limb ischemia of angiogenesis and arteriogenesis were significantly increased only in the ischemic limb; and B) following two days treatment in healthy humans mitochondrial oxygen efficiency was increased causing a reduction in oxygen cost during exercise. The hypothesis of this proposal is that in T2D+PAD subjects with IC, regular consumption of a high nitrate supplement which raises plasma nitrite, in conjunction with 12 weeks of supervised exercise training at the limb ischemic threshold (SET) will produce a greater clinical benefit (increases in COT and PWT) relative to a group with the same exercise training but supplemented with placebo (PET). In order to adequately develop power and execute a larger study, the following specific aims will explore our hypothesis using 24 individuals (12 per group) with T2D+PAD and IC: Specific Aim 1: To determine group differences in the change in COT, PWT and VO2peak during a maximal graded exercise test, following 12 weeks of supervised exercise training. Specific Aim 2: To determine group differences in the change in (a) tissue oxygenation (by NIRS) and (b) NO-derived species (plasma and RBC nitrite, nitrate, nitrosothiols, plasma nitrotyrosine), cGMP during a maximal graded exercise test, and (c) vascular function at rest (endothelial function), calf blood flow (plethysmography). Specific Aim 3a: To determine group differences in the change in gastrocnemius muscle for (a) angiogenesis (capillaries per unit area and per muscle fiber, endothelial cells with surrounding pericytes, relative fraction of type I, versus type II fibers). If differences exist we will look for changes in cell proliferation (PCNA) nd apoptosis (TUNEL); (b) oxidative capacity (citrate synthase activity); (c) mitochondrial volume and density; (d) mitochondrial oxygen efficiency (respiratory control ratio, the ratio of ATP phosphorylation rate per oxygen consumption rate (P/O ratio), and maximal rate of ATP production).