Aerobic Exercise Attenuates Obesity-Associated Vascular Dysfunction via Restoration of Perivascular Adipose Tissue Homeostasis in Mice

Article information

Int Neurourol J. 2025;29(Suppl 1):S35-S43

Publication date (electronic) : 2025 July 31

doi : https://doi.org/10.5213/inj.2550142.071

Meng Li^1,2, Wei Li^,1,3, Yaxiong Zheng¹, Wooyeon Jo¹, Soyoon Lee¹, Seyeon Jang¹, Minseo Kim¹, Hyung Min Kim⁴, Gil-Saeng Jeong⁴, Kee K. Kim⁵, Sang Ki Lee^,1

¹Department of Sport Science, Chungnam National University, Daejeon, Korea

²Department of Rehabilitation Medicine, Zhongguancun Hospital, Beijing, China

³Exercise and Metabolism Research Center, College of Physical Education and Health Sciences, Zhejiang Normal University, Jinhua, China

⁴College of Pharmacy, Chungnam National University, Daejeon, Korea

⁵Department of Biochemistry, Chungnam National University, Daejeon, Korea

Corresponding author: Sang Ki Lee Department of Sport Science, College of Natural Science, Chungnam National University, 99 Daehak-ro, Yuseong-gu, Daejeon 34134, Korea Email: nicelsk@cnu.ac.kr

Co-corresponding author: Wei Li Exercise and Metabolism Research Center, College of Physical Education and Health Sciences, Zhejiang Normal University, Jinhua, China Email: ty1986@zjnu.edu.cn

Received 2025 April 30; Accepted 2025 July 1.

Abstract

• HIGHLIGHTS

- Aerobic exercise training reduces body weight and perivascular adipose tissue (PVAT) lipid accumulation in obese mice.

- Aerobic exercise enhances endothelium-dependent relaxation in the aorta, with and without PVAT.

- Aerobic exercise modulates inflammatory cytokines, adipokine balance and AMPK/Akt/eNOS axis in both of aorta and PVAT.

- Aerobic exercise attenuates vascular dysfunction by restoring PVAT homeostasis.

INTRODUCTION

Adipose tissue, primarily composed of adipocytes, is broadly categorized into white adipose tissue (WAT) and brown adipose tissue (BAT). WAT serves as the body’s main energy reservoir and provides mechanical protection for internal organs. However, excessive WAT accumulation is strongly associated with metabolic disorders, including obesity, type 2 diabetes, and cardiovascular disease [1]. In contrast, BAT plays key roles in thermogenesis and energy expenditure and is considered beneficial for metabolic health [2].

In addition to WAT and BAT, ectopic fat depots such as perivascular adipose tissue (PVAT) have emerged as important regulators of vascular function. PVAT surrounds most large arteries and exerts paracrine effects on the vasculature by releasing vasoactive factors. Under physiological conditions, PVAT exhibits anticontractile properties that help regulate vascular tone [3]. However, in obesity and metabolic syndrome, PVAT undergoes structural and functional changes, including adipocyte hypertrophy, inflammatory cytokine secretion, and impaired vasodilatory signaling, ultimately promoting vascular dysfunction [4-6].

Recent research has indicated that obesity-induced PVAT inflammation increases proinflammatory cytokine expression and promotes the infiltration of immune cells, including macrophages [7]. Moreover, PVAT inflammation has been linked to vascular diseases such as hypertension and atherosclerosis [8], in which it diminishes the anticontractile effects on small arteries [9, 10].

AMP-activated protein kinase (AMPK) is a central regulator of metabolic and vascular homeostasis and modulates endothelial nitric oxide synthase (eNOS) activity. AMPK expression and activity are known to be reduced in obesity and vascular dysfunction [11-13]. Although several studies have shown that exercise activates AMPK and eNOS in endothelial cells and skeletal muscle, little is known about the effects of exercise on AMPK/eNOS signaling within PVAT in obesity.

Regular aerobic exercise is a well-established strategy for improving vascular function and reducing inflammation in animal models and humans with obesity [13-15]. Several animal studies have demonstrated that exercise promotes endothelium-dependent relaxation (EDR) and reduces oxidative stress, partly by activating AMPK and eNOS [16]. However, the specific effects of aerobic exercise on PVAT-induced vascular dysfunction, particularly in the context of high-fat diet-induced obesity, remain poorly understood.

While several studies have demonstrated that exercise improves endothelial function through the activation of AMPK and eNOS, these findings have primarily focused on the vascular wall itself [14, 15], with limited attention given to PVAT. Very few studies have comprehensively addressed how exercise affects vascular function through the integrated roles of the vascular wall and the surrounding PVAT, representing a critical gap in our understanding of the vascular benefits of exercise in obesity.

Therefore, the purpose of this study was to investigate whether aerobic exercise training could ameliorate vascular dysfunction by restoring PVAT homeostasis in a mouse model of high-fat diet-induced obesity.

MATERIALS AND METHODS

Quantitative Real-Time Polymerase Chain Reaction

Total RNA was extracted from frozen aorta and PVAT tissues using TRIzol reagent (Life Technologies, USA), followed by chloroform extraction and ethanol precipitation. RNA concentration and purity were determined using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, USA). Complementary DNA (cDNA) was synthesized using oligo(dT)15 primers (Takara Bio Inc., Japan) and reverse transcriptase (NEB, China) according to the manufacturer’s instructions. Quantitative real-time polymerase chain reaction (PCR) was performed using SYBR Green Master Mix on an Applied Biosystems 7500 Real-Time PCR System (Thermo Fisher Scientific). Each reaction (25 μL) contained cDNA, gene-specific primers (TsingKe, China), ROX dye, and RNase-free water. The PCR cycling conditions included initial denaturation at 95°C for 15 minutes, followed by 40 cycles of 95°C for 10 seconds and 60°C for 30 seconds, with a final melting curve analysis. Primer sequences and melting temperatures are listed in Table 1.

Table 1.

Primer sequences used for real-time polymerase chain reaction

RESULTS

Aerobic Exercise Training Inhibits Body Weight Gain Without Altering Energy Intake

Body weight changes are presented in Fig. 1A. Following 12 weeks of a high-fat diet, 8 weeks of aerobic exercise training significantly inhibited body weight gain compared to both the C group (30.84 ± 0.20 g vs. 28.68 ± 0.06 g) and the HF group (30.84 ± 0.20 g vs. 37.04 ±0.16 g), without significantly altering energy intake (Fig. 1B).

Fig. 1.

Aerobic exercise training inhibited body weight gain without altering energy intake in high-fat diet-induced mice. (A) Body weight. (B) Energy intake. C, chow diet control; C+E, chow diet with exercise; HF, high-fat diet; HF+E, high-fat diet with exercise. Data are presented as mean±standard error of the mean. *P<0.05, C vs. C+E; ^‡P<0.05, C vs. HF; ^#P<0.05, HF vs. HF+E.

Aerobic Exercise Training Reduces Lipid Droplet Content in PVAT

The effects of aerobic exercise training on lipid droplet content in PVAT were examined (Fig. 2). Compared to the C group (187.10 ± 5.38 μm²), the HF group (206.98 ± 1.04 μm²) exhibited a significant increase in lipid droplet content (Fig. 2B and C). In high-fat diet-fed mice, aerobic exercise training (HF+E, 198.40 ±1.45 μm²) significantly reduced PVAT adipocyte size compared to the HF group (Fig. 2B and C).

Fig. 2.

Aerobic exercise training reduced lipid droplet content in the perivascular adipose tissue (PVAT) of high-fat diet-induced mice. (A) Thoracic aorta and PVAT. (B) Lipid droplets in PVAT. (C) Quantification of lipid droplets in PVAT by planimetry. C, chow diet control; C+E, chow diet with exercise; HF, high-fat diet; HF+E, high-fat diet with exercise. Data are presented as mean±standard error of the mean. ^‡P<0.05, C vs. HF; ^#P<0.05, HF vs. HF+E. Hematoxylin and eosin images: ×100 (A), ×400 (B); scale bars: 100 μm (A), 50 μm (B).

Aerobic Exercise Improves EDR in the Aorta With and Without PVAT

The effects of aerobic exercise training on EDR in the thoracic aorta were investigated (Fig. 3). The HF group exhibited significantly reduced EDR in response to 10 –5 M acetylcholine compared to the C group, both with PVAT (40.10±0.58 vs. 98.94±0.40) and without PVAT (47.92±0.44 vs. 96.37±0.13) (Fig. 3A and B). However, aerobic exercise training significantly improved EDR in the aorta compared to the HF group, both with (59.31 ±0.71 vs. 40.10 ±0.58) and without PVAT (65.27 ±1.32 vs. 47.92 ± 0.44) (Fig. 3A and B).

Fig. 3.

Aerobic exercise training improved endothelial-dependent relaxation (EDR) in the aorta with and without perivascular adipose tissue (PVAT) in high-fat diet-induced mice. (A) EDR with PVAT. (B) EDR without (w/o) PVAT. C, chow diet control; C+E, chow diet with exercise; HF, high-fat diet; HF+E, high-fat diet with exercise. Data are presented as mean±standard error of the mean. ^‡P<0.05, C vs. HF; ^#P<0.05, HF vs. HF+E.

Aerobic Exercise Suppresses High-Fat Diet-Induced Proinflammatory Cytokine Expression and Promotes Anti-Inflammatory Cytokine Expression in the Aorta and PVAT

mRNA expression levels for inflammatory cytokines (tumor necrosis factor alpha [TNF-α], interleukin 1β, monocyte chemoattractant protein 1 [MCP-1], intercellular adhesion molecule 1 [ICAM-1], and leptin) and an anti-inflammatory adipokine (adiponectin) were analyzed in the aorta and PVAT (Fig. 4). Compared to the C group, the HF group displayed significant increases in proinflammatory cytokine (TNF-α, 35.91 ± 1.92 vs. 0.98 ±0.01) and adipokine (leptin, 9.39 ±0.06 vs. 1.07 ±0.06) expression (Fig. 4A–E) but a decrease in anti-inflammatory adipokine (adiponectin, 0.05±0.01 vs. 0.86±0.04) expression (Fig. 4F). In contrast, aerobic exercise training significantly reduced proinflammatory cytokine expression (TNF-α, 12.98 ±2.20 vs. 35.91 ±1.92) and increased anti-inflammatory adipokine mRNA levels (adipokine, 0.05±0.01 vs. 0.28±0.05) in both the aorta (Fig. 4A–F) and PVAT (Fig. 4G–L), compared to the HF group.

Fig. 4.

Aerobic exercise improved levels of proinflammatory and anti-inflammatory cytokines in the aorta and perivascular adipose tissue (PVAT) of high-fat diet-induced mice. The images depict relative mRNA levels of tumor necrosis factor alpha (TNF-α), interleukin (IL)-1β, monocyte chemoattractant protein 1 (MCP-1), intercellular adhesion molecule 1 (ICAM-1), leptin, and adiponectin in the aorta (A–F) and PVAT (G–L). C, chow diet control; C+E, chow diet with exercise; HF, high-fat diet; HF+E, high-fat diet with exercise. Data are presented as mean±standard error of the mean. *P<0.05, C vs. C+E; ^‡P<0.05, C vs. HF; ^#P<0.05, HF vs. HF+E.

Aerobic Exercise Improves Phosphorylation of AMPK/Akt/eNOS in the Aorta and PVAT

Phosphorylation levels of AMPK, Akt, and eNOS were measured in the aorta and PVAT (Fig. 5). The HF group exhibited significantly reduced phosphorylation of AMPK (0.38±0.01 vs. 1.00±0.00), Akt (0.59±0.03 vs. 1.00±0.00), and eNOS (0.34±0.01 vs. 1.00±0.00) in the aorta (Fig. 5A) and PVAT (AMPK, 0.22±0.01 vs. 1.00±0.00; Akt, 0.49±0.02 vs. 1.00±0.00; eNOS, 0.52 ±0.02 vs. 1.00 ±0.00) (Fig. 5B) compared to the C group. However, aerobic exercise training significantly restored the phosphorylation of these signaling molecules in both the aorta (AMPK, 0.61 ±0.04 vs. 0.38 ±0.01; Akt, 0.81 ±0.08 vs. 0.59 ± 0.03; eNOS, 0.74 ±0.01 vs. 0.34 ±0.01) (Fig. 5A) and PVAT (AMPK, 0.76 ±0.07 vs. 0.22 ±0.01; Akt, 0.87 ±0.04 vs. 0.49 ± 0.02; eNOS, 0.72 ±0.00 vs. 0.52 ±0.02) (Fig. 5B) compared to the HF group.

Fig. 5.

Aerobic exercise promoted phosphorylation of AMP-activated protein kinase (AMPK)/Akt/endothelial nitric oxide synthase (eNOS) in the aorta and perivascular adipose tissue (PVAT) of high-fat diet-induced mice. The images depict western blotting and quantification of the expression and phosphorylation of AMPK, AKT, and eNOS in the aorta (A) and PVAT (B). p-AMPK, phospho-AMPK; p-AKT, phospho-AKT; p-eNOS, phospho-eNOS; C, chow diet control; C+E, chow diet with exercise; HF, high-fat diet; HF+E, high-fat diet with exercise. Data are presented as mean±standard error of the mean. *P<0.05, C vs. C+E; ^‡P<0.05, C vs. HF; ^#P<0.05, HF vs. HF+E.

DISCUSSION

This study demonstrates that aerobic exercise training exerts beneficial effects on vascular function and inflammation in both the thoracic aorta and PVAT in mice with obesity induced by a high-fat diet. The C57BL/6J mice, known for their susceptibility to diet-induced obesity [16], exhibited approximately 30% weight gain after 12 weeks of high-fat diet feeding, confirming successful establishment of the obesity model. Aerobic exercise reduced body weight and fat mass, likely through increased energy expenditure, improved lipid oxidation, and regulation of obesity-related gene expression (Fig. 1). While body weight is widely used as a key obesity indicator, the exercise-induced effects observed in this study may reflect improved metabolic function rather than changes in size alone [18, 19].

Regarding vascular function, the present findings indicate that PVAT dysfunction exacerbates impairment in EDR, particularly in the context of obesity. Acetylcholine-induced vasodilation was more severely impaired in aortic segments with intact PVAT, highlighting the pathological role of PVAT dysfunction (Fig. 3). Aerobic exercise significantly improved this vasodilatory response, accompanied by increased phosphorylation of eNOS and its upstream regulator Akt in both the aorta and PVAT (Figs. 3A, B, and 5). These results suggest that the beneficial vascular effects of exercise are mediated through the Akt/eNOS signaling pathway.

Previous studies have demonstrated that a chronic high-fat diet suppresses eNOS expression and activity [20, 21], while exercise restores these parameters in PVAT [22-24]. Consistent with this mechanism, treadmill training in the current study increased both p-eNOS and p-Akt levels in vascular tissues, supporting the role of this pathway in improving PVAT-mediated vascular function.

In addition to enhancing signaling activity, exercise training effectively reduced inflammation in PVAT [25]. As an active endocrine organ, PVAT secretes numerous cytokines that modulate vascular tone [9]. In this study, aerobic exercise suppressed the expression of proinflammatory markers, including TNF-α, ILβ, MCP-1, ICAM-1, and leptin, while promoting the expression of the anti-inflammatory adipokine adiponectin in both the aorta and PVAT (Fig. 4). Histological analysis revealed smaller adipocytes and reduced lipid droplet content in the PVAT of mice subjected to exercise (Fig. 2B and C). Moreover, the morphological characteristics of PVAT, including circular nuclei and multilocular lipid droplets, resembled BAT; this finding aligns with reports that aortic PVAT comprises mixed white and brown adipocytes [26, 27].

Leptin, which is often elevated in obesity, contributes to atherosclerosis by promoting monocyte chemotaxis, foam cell formation, and vascular smooth muscle cell proliferation [28]. The observed increase in leptin mRNA in the PVAT of obese mice — as well as its reduction following the exercise regimen — suggests a local, PVAT-specific effect on vascular dysfunction (Fig. 4E and K). Previous studies have also indicated that PVAT-derived leptin may act independently of systemic leptin levels [29, 30] and that inhibition of leptin signaling in PVAT improves endothelial function in the context of obesity [31].

Conversely, adiponectin is known to exert anti-inflammatory and antioxidative effects and plays a protective role in vascular physiology [32]. In this study, exercise increased adiponectin expression, likely contributing to enhanced phosphorylation of AMPK in PVAT. AMPK activation improves vascular homeostasis by regulating lipid metabolism, inflammation, and cellular signaling [33]. Moreover, AMPK phosphorylation in PVAT may reflect enhanced metabolic capacity, aligning with previous reports of AMPK upregulation following exercise, although these findings were initially observed in skeletal muscle [34].

In this study, aerobic exercise significantly increased AMPK phosphorylation in both the thoracic aorta and PVAT (Fig. 5), indicating tissue-wide metabolic activation. Notably, the magnitude of AMPK phosphorylation was greater in PVAT, suggesting higher metabolic responsiveness in adipose tissue compared to vascular tissue. In the aorta, AMPK activation may have contributed primarily to endothelial homeostasis by promoting eNOS phosphorylation and nitric oxide bioavailability, as previously reported [35]. In contrast, in PVAT, AMPK signaling appears to regulate not only vascular tone through paracrine factors but also adipocyte metabolism and inflammation [33, 36]. This differential role highlights the tissue-specific actions of AMPK in mediating both structural and functional recovery of the vascular environment in obesity.

Collectively, these findings indicate that aerobic exercise training ameliorates PVAT-induced vascular dysfunction in obese mice by reducing lipid accumulation in PVAT, alleviating PVAT inflammation, restoring adipokine balance, and activating the AMPK–Akt–eNOS axis. These mechanisms highlight the therapeutic potential of exercise as an effective nonpharmacological strategy to protect against obesity-related vascular complications through PVAT modulation.

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