Exercise performance and peripheral vascular insufficiency improve with AMPK activation in high-fat diet-fed mice

Abstract

Intermittent claudication is a form of exercise intolerance characterized by muscle pain during walking in patients with peripheral artery disease (PAD). Endothelial cell and muscle dysfunction are thought to be important contributors to the etiology of this disease, but a lack of preclinical models that incorporate these elements and measure exercise performance as a primary end point has slowed progress in finding new treatment options for these patients. We sought to develop an animal model of peripheral vascular insufficiency in which microvascular dysfunction and exercise intolerance were defining features. We further set out to determine if pharmacological activation of 5'-AMP-activated protein kinase (AMPK) might counteract any of these functional deficits. Mice aged on a high-fat diet demonstrate many functional and molecular characteristics of PAD, including the sequential development of peripheral vascular insufficiency, increased muscle fatigability, and progressive exercise intolerance. These changes occur gradually and are associated with alterations in nitric oxide bioavailability. Treatment of animals with an AMPK activator, R118, increased voluntary wheel running activity, decreased muscle fatigability, and prevented the progressive decrease in treadmill exercise capacity. These functional performance benefits were accompanied by improved mitochondrial function, the normalization of perfusion in exercising muscle, increased nitric oxide bioavailability, and decreased circulating levels of the endogenous endothelial nitric oxide synthase inhibitor asymmetric dimethylarginine. These data suggest that aged, obese mice represent a novel model for studying exercise intolerance associated with peripheral vascular insufficiency, and pharmacological activation of AMPK may be a suitable treatment for intermittent claudication associated with PAD.

Keywords: 5′-AMP-activated protein kinase; exercise; intermittent claudication; nitric oxide; obesity.

Fig. 1.

Hindlimb skeletal muscle perfusion is reduced in male C57BL/6 mice fed a high-fat diet (HFD). A: body mass. ND, normal diet. B: oral glucose tolerance testing after 46 wk of ND (n = 34) and HFD (n = 33). C: representative contrast-mode ultrasound images of the medial aspect of the lower hindlimb of ND- and HFD mice. D: peak enhancement (PE) is the maximum-minimum video intensity and is an indicator of blood volume. E: average PE in the hindlimb of ND-fed (n = 13–18) and HFD-fed (n = 21–35 except at 20 wk, where n = 12) mice. Only half of the HFD-fed mice were measured at the 20-wk time point. AU, arbitrary units. F: cardiac function in ND-fed (n = 11) and HFD-fed (n = 10) mice. HR, heart rate; SV, stroke volume; CO, cardiac output; EF, ejection fraction; FS, fractional shortening; LV mass, left ventricular (LV) mass; LVEDV, LV end-diastolic volume; LVPWT, LV posterior wall thickness. Data are presented as means ± SE. Data were analyzed with two-tailed, independent t-tests between ND and HFD at each time point. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 2.

Exercise capacity is impaired in male C57BL/6 mice fed a HFD. A: voluntary wheel running activity during a 1-h timed test in ND- and HFD-fed mice. B: rest time (1-min periods with zero counts). C: average speed calculated when mice were engaged with the wheel. Sample sizes for ND-fed mice were as follows: 17 wk (n = 16) and 28 wks (n = 18); sample sizes for HFD-fed mice were as follows: 17 wk (n = 32) and 28 wk (n = 29). D: treadmill exhaustion time in ND- and HFD-fed mice. Sample sizes for ND-fed mice were as follows: 10–29 wk (n = 18) and 35 wk (n = 16); sample sizes for HFD-fed mice were as follows: 10–15 wk (n = 33), 19 wk (n = 17), 29 wk (n = 27), and 35 wk (n = 26). Only half of the HFD-fed mice were tested at 19 wk of HFD. Data are presented as means ± SE. Data were analyzed with two-tailed, independent t-tests between ND and HFD at each time point. **P < 0.01; ***P < 0.001.

Fig. 3.

Obesity-induced reductions in hindlimb perfusion are independent of large vessel atherosclerosis. A and B: representative images of atherosclerosis in the aorta (A) and femoral artery (B) from ND-fed (n = 5), HFD-fed (n = 6), and apolipoprotein E gene (ApoE)-deficient (ApoE^−/−) mice (n = 5). Mice were assessed after 49–73 wk of HFD, and ApoE^−/− mice were 65 wk old. C: C57BL/6 and ApoE^−/− mice were also tested for skeletal muscle perfusion (n = 5–7 mice/group except at the age of 4 mo, where n = 3 mice/group). D: treadmill exercise capacity in C57BL/6 and ApoE^−/− mice (n = 14–20 mice/group). Data are presented as means ± SE. Data were analyzed with independent t-tests between C57BL/6 and ApoE^−/− mice at each time point. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 4.

Microvascular dysfunction in skeletal muscle and nitric oxide (NO) bioavailability contribute to vascular insufficiency in aged, obese mice. A: representative micro-computed tomography (CT) images of AltaBlu-filled gastrocnemius/plantaris muscles delineating blood vessels after 73 wk of ND (n = 7) or HFD (n = 6) in male C57BL/6 mice. Scale bar = 200 μm. B: frequency distribution of vessel radius. C: frequency distribution of vessel branching. D: representative ×40 medial gastrocnemius muscle sections depicting capillary density (CD31⁺: green; laminin: red). Scale bar = 100 μm. Insets show higher-magnification images. E: quantification of capillary density. F and G: plasma total nitrate and nitrite concentration (F) and plasma asymmetric dimethylarginine (ADMA; G) after 46 wk of HFD (n = 8 mice/group). Data are presented as means ± SE. Data were analyzed with two-tailed, independent t-tests between ND and HFD. *P < 0.05; ***P < 0.001.

Fig. 5.

R118 activates 5′-AMP-activated protein kinase (AMPK) through an indirect mechanism of action. A: R118 does not increase AMPK activity in vitro. Kinase activity was measured in the presence or absence of 1 μM AMP for α₁β₁γ₁ and 10 μM for α₂β₁γ₁ (n = 2/group). B: R118 inhibits mitochondrial respiration in a dose-dependent fashion. HepG2 cells were incubated with 0.05% DMSO or different concentrations of R118, and the O₂ consumption rate (OCR) was measured in duplicate using a XF24 Seahorse instrument. All data are normalized to the time point before the addition of R118. C: R118 inhibits mitochondrial respiration by inhibiting complex I. The OCR was measured in 5 μg of purified mouse liver mitochondria in triplicate in the basal state with the addition of a complex I inhibitor [rotenone (Ro)], a stimulator of complex II [succinate (Suc)], an inhibitor of complex II [antimycin A (Ant)], and a stimulator of complex IV [ascorbate/N,N,N′,N′-tetramethyl-p-phenylenediamine (As/T)]. Sample sizes were as follows: DMSO (n = 3), 0.3 μM R118 (n = 4), and 3.3 μM R118 (n = 4). D: NADH oxidation was blunted by R118. Purified liver mitochondrial lysates were analyzed spectrophotometrically in duplicate for the conversion of NADH to NAD⁺ in the presence of 2 mM NADH. ***P < 0.001 vs. DMSO.

Fig. 6.

R118 activates AMPK and endothelial NO synthase (eNOS) in vitro and in vivo. A: representative Western blot showing phosphorylated (p)AMPK (Thr¹⁷²) activation in gastrocnemius muscle 60 min after oral dosing of R118 at 2.5, 5, and 10 mg/kg body mass (n = 4 mice/group) in 11.5-wk-old C57BL/6 wild-type mice and quantitation of pAMPK (Thr¹⁷²). B: plasma levels of R118 after acute dosing. Sample sizes at each time point were as follows: n = 4 mice/group except 7 h (n = 3) and 24 h (n = 2). C: human microvascular endothelial cells (HuMECs) were treated with DMSO or R118 at increasing concentrations (9, 27, 82, 244, 733, and 2,220 nM) for 10 min. The triangle represents the increasing concentration of R118. This experiment was replicated two times. D: R118 activates eNOS in the aorta in vivo. Male C57BL/6 mice fed a HFD for 47 wk were dosed orally with R118 or vehicle at 10 mg/kg body mass, and aortas were collected 60 min after dosing (n = 6 mice/group). Western blots of phosphorylation of AMPK (Thr¹⁷²) and eNOS (Ser¹¹⁷⁷) are shown with a quantification of pAMPK (Thr¹⁷²) and p-eNOS (Ser¹¹⁷⁷). IOD, integrated optical density. Data are presented as means ± SE. Data were analyzed with two-tailed, independent t-tests or one-way ANOVA with Holm-Sidak's post hoc test. *P < 0.05; ***P < 0.001 vs. vehicle (Veh).

Fig. 7.

Exercise capacity is improved with R118. Mice were singly housed with a running wheel for 1 h for 3 consecutive days. Sample sizes for groups were as follows: ND (n = 32), HFD untreated (Unt; n = 31), HFD + wheel exercise (n = 16), HFD + low-dose cilostazol (cilostazol-low; n = 33), HFD + high-dose cilostazol (cilostazol-high; n = 17), HFD + AT-1015 (n = 17), HFD + low-dose R118 (R118-low; n = 29), and HFD + high-dose R118 (R118-high; n = 32). A and B: total counts (A) and rest time (B), defined as the number of 1-min intervals without a count. C: average speed was determined only when mice were actively engaged with the wheel. All data were determined and averaged over 3 days. Data are presented as means ± SE. Data were analyzed by one-way ANOVA with repeated measures and Holm-Sidak's post hoc test. *P < 0.05; **P < 0.01; ***P < 0.001 vs. pretreatment (Pre).

Fig. 8.

R118 improves skeletal muscle fatigue after in vivo plantar flexion contractions. The following groups of mice were evaluated: ND (n = 18), HFD-Unt (n = 15), HFD + wheel exercise (n = 15), HFD + cilostazol-high (n = 15), HFD + AT-1015 (n = 16), HFD + R118-low (n = 9), and HFD + R118-high (n = 16). A: maximal isometric torque. B–D: plantar flexion torque was measured during fatiguing contractions in ND, Unt, and wheel-exercised mice (B), R118-treated mice (C), and cilostazol- and AT-1015-treated mice (D). Exercise consisted of 60 plantar flexion contractions via stimulation of the sciatic nerve with percutaneous electrodes at 100 Hz, 200-ms train duration, and 0.15-ms pulse duration. One contraction was elicited every 2 s for 2 min. Data were collected 17–23 wk posttreatment. For maximal isometric torque, data are presented as means +/- SE. For fatigue, only means are presented. The Untreated (Unt) group is regraphed in each Fig. and represents the same data set. Data were analyzed by 1-way or 2-way ANOVA with Holm-Sidak's posthoc. Bars signify contractions that are different from HFD-Unt. *P < 0.05.

Fig. 9.

Skeletal muscle mitochondrial function is improved with R118. Plantaris muscles were collected 18 wk after treatment. A sample size of n = 7–8 was used for all measurements, except the HFD + Wheel (n = 5). Data are presented as mean +/- SE. Data were analyzed by one-way ANOVA with Holm-Sidak's posthoc (Untreated vs. all other treatments). *P < 0.05, ***P < 0.001 vs. HFD-Unt.

Fig. 10.

R118 improves skeletal muscle perfusion at rest and after exercise. Mice were fed a ND (n = 18), HFD-Unt (n = 15), HFD + Wheel (n = 15), HFD + Cilostazol-high (n = 14), HFD + AT-1015 (n = 13), HFD + R118-low (n = 9), or HFD + R118-high (n = 16). Hind limb muscle perfusion was measured with contrast-enhanced ultrasound at rest and after exercise in the same limb. Exercise consisted of 120 plantar flexion contractions via stimulation of the sciatic nerve with percutaneous electrodes at 100 Hz, 200-ms train duration, and 0.15-ms pulse duration. One contraction was elicited every 2 s for 2 min, followed by a 1-min rest and a second set of 60 contractions. Data were collected 17–22 wk posttreatment. A: representative peak enhancement images before (rest) and after fatiguing muscle contractions (exercise). B and C: peak enhancement at rest (B) and after exercise (C). Data are presented as means ± SE. Data were analyzed by one-way ANOVA with Holm-Sidak's post hoc test. *P < 0.05; **P < 0.01; ***P < 0.001 vs. HFD-Unt.

Fig. 11.

R118 increases the number of detectable small vessels and branch points per vessel length in skeletal muscle, and this is related to NO bioavailability. The left leg was subjected to 120 plantar flexion contractions (exercise), and the contralateral limb served as a control (rest). There was no effect of exercise or an interaction, so data presented are the main effects of treatment (vessel radius: P = 0.034; vessel branching: P = 0.004). A: representative micro-CT images of the gastrocnemius muscle from the unexercised leg from the ND (n = 14), HFD-Unt (n = 12), HFD + cilostazol-high (n = 12), HFD + R118-low (n = 12), and HFD + R118-high (n = 12) groups. Muscles were collected 27–29 wk posttreatment. AT-1015-treated mice were not tested. Scale bar = 2 mm. B: mean number of small vessels (≤12 μm in radius). C: vessels with multiple branch points (≥3 branch points/mm). D and E: plasma nitrate and nitrite (D) and plasma ADMA (E) from the ND (n = 18), HFD-Unt (n = 18), HFD + cilostazol-high (n = 14), HFD + AT-1015 (n = 18), HFD + R118-low (n = 18), and HFD + R118-high (n = 18) groups. Plasma samples were collected 25 wk posttreatment. Data are presented as means ± SE. Data were analyzed by one- or two-way ANOVA with Holm-Sidak's post hoc test. *P < 0.05; **P < 0.01; ***P < 0.001 vs. HFD-Unt.

Fig. 12.

Similarities between patients with peripheral artery disease (PAD) and aged, obese mice and the efficacy of R118, cilostazol, and AT-1015 in this animal model. All data represent chronic treatment with different compounds. PDE3, phosphodiesterase 3; 5-HT, serotonin; N/A, not applicable.