Objective-The regulation of AMP-activated protein kinase (AMPK) is implicated in vascular biology because AMPK can phosphorylate endothelial NO synthase (eNOS). In this study, we investigate the regulation of the AMPK-eNOS pathway in vascular endothelial cells (ECs) by shear stress and the activation of aortic AMPK in a mouse model with a high level of voluntary running (High-Runner). Methods and Results-By using flow channels with cultured ECs, AMPK Thr172 phosphorylation was increased with changes of flow rate or pulsatility. The activity of LKB1, the upstream kinase of AMPK, and the phosphorylation of eNOS at Ser1179 were concomitant with AMPK activation responding to changes in flow rate or pulsatility. The blockage of AMPK by a dominant-negative mutant of AMPK inhibited shear stress-induced eNOS Ser1179 phosphorylation and NO production. Furthermore, aortic AMPK activity and level of eNOS phosphorylation were significantly elevated in the aortas of High-Runner mice. Conclusions-Our results suggest that shear stress activates AMPK in ECs, which contributes to elevated eNOS activity and subsequent NO production. Hence, AMPK, in addition to serving as an energy sensor, also plays an important role in regulating vascular tone. Key Words: endothelium Ⅲ AMPK Ⅲ nitric oxide synthase Ⅲ shear stress Ⅲ exercise E ndothelium-derived NO can enhance vascular functions, including vessel relaxation, survival of vascular endothelial cells (ECs), inhibition of platelet aggregation, and attenuation of leukocyte infiltration. 1,2 Impaired NO bioavailability has been suggested as one of the earliest pathophysiological events preceding endothelial dysfunction and contributing to atherosclerosis. 3,4 Shear stress is an important physiological stimulus that enhances the production of NO by ECs. 2,5 An increase in shear stress such as in exercise augments the EC-mediated bioavailability of NO. 6 Endothelial NO synthase (eNOS), the key enzyme for NO production in ECs, is tightly regulated not only at the transcriptional level but also by several post-translational mechanisms. The enhanced phosphorylation of Ser1179 of bovine eNOS (Ser1177 in humans) leads to increased eNOS activity. Mounting evidence has shown that shear stress enhances the phosphorylation of Ser1177/1179. 7-9 Use of the phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin and LY 294002 has demonstrated that Akt phosphorylates eNOS Ser1177/1179 in response to shear stress. 7,8 However, dominant-negative mutants of Akt were unable to block the shear stress-stimulated Ser1179 phosphorylation. 9 Further, H89, a protein kinase A (PKA) inhibitor, and an adenovirusexpressing PKA inhibitor (PKI) blocked the eNOS Ser1179 phosphorylation, which indicates the involvement of PKA. 9 -12 Functioning as a metabolic master switch, AMP-activated protein kinase (AMPK) senses and regulates the cellular energy status in various cell types. AMPK is activated by several physiological and pathological stresses such as exercise, hypoxia, and nutrient depletion that result in incre...

The response to uniform selection may occur in alternate ways that result in similar performance. We tested for multiple adaptive solutions during artificial selection for high voluntary wheel running in laboratory mice. At generation 43, the four replicate high runner (HR) lines averaged 2.85-fold more revolutions per day as compared with four non-selected control (C) lines, and females ran 1.11-fold more than males, with no sex-by-linetype interaction. Analysis of variance indicated significant differences among C lines but not among HR for revolutions per day. By contrast, average speed varied significantly among HR lines, but not among C, and showed a sex-by-linetype interaction, with the HR/C ratio being 2.02 for males and 2.45 for females. Time spent running varied among both HR and C lines, and showed a sex-by-linetype interaction, with the HR/C ratio being 1.52 for males but only 1.17 for females. Thus, females (speed) and males (speed, but also time) evolved differently, as did the replicate selected lines. Speed and time showed a trade-off among HR but not among C lines. These results demonstrate that uniform selection on a complex trait can cause consistent responses in the trait under direct selection while promoting divergence in the lower-level components of that trait.

Dysfunction of the hypothalamic-pituitary-adrenal axis resulting in elevated baseline glucocorticoid concentrations is a hallmark of stress-related human anxiety and affective disorders, including depression. Mice from four replicate lines bred for high voluntary wheel running (HR lines) run almost three times as much as four non-selected control (C) lines, and exhibit two fold elevated baseline circulating corticosterone levels throughout the 24 h cycle. Although elevated baseline CORT may be beneficial for high locomotor activity, chronic elevations can have deleterious effects on multiple systems, and may predispose for affective disorders. Because stressful events often precede a depressive bout, we quantified depressive-like behavior in the forced-swim (FST; generation 41) and tail-suspension tests (TST; generation 47) in HR and C mice that had wheel access for 6 days and then were deprived of wheels on day seven prior to the FST or TST. Male HR spent significantly more time immobile in the FST than C, suggesting that HR males have a predisposition for depression-like behavior. Both male and female HR (generation 43) were more active than same-sex controls in both wheel running and home-cage activity across 22 h (pooling the sexes, HR/C = 2.28 and 2.66, respectively).

SUMMARYVoluntary activity is a complex trait, comprising both behavioral (motivation, reward) and anatomical/physiological (ability) elements. In the present study, oxygen transport was investigated as a possible limitation to further increases in running by four replicate lines of mice that have been selectively bred for high voluntary wheel running and have reached an apparent selection limit. To increase oxygen transport capacity, erythrocyte density was elevated by the administration of an erythropoietin (EPO) analogue. Mice were given two EPO injections, two days apart, at one of two dose levels (100 or 300gkg -1 and 300gkg -1 dose levels (overall mean of 4.5gdl -1 increase). EPO treatment significantly increased V O2,max by ~5% in both the HR and C lines, with no dose ϫ line type interaction. However, wheel running (revolutions per day) did not increase with EPO treatment in either the HR or C lines, and in fact significantly decreased at the higher dose in both line types. These results suggest that neither [Hb] per se nor V O2,max is limiting voluntary wheel running in the HR lines. Moreover, we hypothesize that the decrease in wheel running at the higher dose of EPO may reflect direct action on the reward pathway of the brain.Key words: artificial selection, central limitation, experimental evolution, maximum metabolic rate, oxygen transport, peripheral limitation, respiratory exchange ratio, selection limit, symmorphosis. THE JOURNAL OF EXPERIMENTAL BIOLOGY 511Erythropoietin elevates V O 2 ,max in mice aerobically supported exercise has also increased in the HR lines, as demonstrated by increased endurance (Meek et al., 2009) and increases maximum oxygen consumption [V O2,max (Swallow et al., 1998b;Rezende et al., 2005;Rezende et al., 2006a)] during forced treadmill exercise. However, the trait, or group of traits, that is limiting to even higher levels of wheel running in the HR lines remains an open question. Results to date indicate that glycogen depletion during nightly running is not responsible for the limitation (Gomes et al., 2009) nor are the HR mice limited by their ability to process enough energy to support the increased energy demands of running (Koteja et al., 1999;Koteja et al., 2001;Vaanholt et al., 2007a;Rezende et al., 2009).One of the main predictors of endurance-running ability is wholeorganism aerobic capacity (Wagner, 1996;Bassett and Howley, 2000;Lucia et al., 2001;Calbet et al., 2006;Noakes, 2007;Levine, 2008;Noakes, 2009). All else being equal, the higher the maximum aerobic capacity, the higher the maximum sustainable running speed. Maximum oxygen consumption defines the upper limits of the cardiovascular/respiratory system and of aerobic ability in general (for a review, see Levine, 2008). The ultimate determinant of organismal aerobic capacity occurs at the 'sink' of tissue oxygen consumption but all of the sequential steps of oxygen transport, from ventilatory convection in the lungs to oxygen diffusion in the peripheral tissues, contribute to V O2,max (Taylor an...