Voluntary exercise eliminates maternal gestational hypertension-induced hypertensive response sensitization to post-weaning high-fat diet in male adult offspring

Abstract

Background:

Exercise has profound effects on cardiovascular function and metabolism in both physiological and pathophysiological states. The present study tested whether voluntary exercise would protect male offspring against maternal gestational hypertension-induced hypertensive response sensitization (HTRS) elicited by post-weaning high-fat diet (HFD).

Methods and Results:

On low-lard-fat diet offspring of both normotensive (NT) and hypertensive (HT) dams had comparable resting blood pressure (BP), but HFD feeding elicited an enhanced increase in BP (i.e., HTRS) in sedentary offspring of HT dams when compared to sedentary offspring of NT dams. The HFD fed sedentary offspring of HT dams displayed greater sympathetic activity, enhanced pressor responses to centrally administered angiotensin II or leptin, and greater mRNA expression of proinflammatory cytokines, leptin, and a marker of blood-brain barrier leakage in the hypothalamic paraventricular nucleus. The enhanced BP and central sympathetic activity in HFD fed sedentary offspring of HT dams were significantly reduced by exercise but fell only to levels comparable to HFD fed exercising offspring of NT dams. HFD-induced increases in plasma interleukin-6 and sympathetic activity and greater pressor responses to central tumor necrosis factor-α in offspring from both NT and HT dams were also maintained after exercise. Nevertheless, exercise had remarkably beneficial effects on metabolic and autonomic function, brain reactivity to angiotensin II and leptin and gene expression of brain prohypertensive factors in all offspring.

Conclusions:

Voluntary exercise plays a beneficial role in preventing maternal hypertension-induced HTRS and that this is associated with attenuation of enhanced brain reactivity and centrally driven sympathetic activity.

Graphical Abstract

Introduction

Hypertension is a major risk factor for heart disease, renal failure and stroke that affects more than 31% of adults worldwide.¹ Both genetic and lifetime environmental factors have important influences on blood pressure (BP). It has been shown that prenatal insults can predispose offspring to cardiometabolic dysfunction, and that physiological challenges or environmental stressors encountered later in life such as the pressor agent angiotensin (ANG) II or high-fat diet (HFD), act as “second-hits” triggering frank disease in the prenatally programmed offspring.^2–4 In this context, we previously demonstrated that maternal gestational hypertension in rats induces latent hypertensive response sensitization (HTRS) that can be elicited by post-weaning HFD in both male and female offspring.^{5, 6}

Physical exercise has been demonstrated to be a non-pharmacological and effective approach for increasing protection against obesity-related cardiometabolic disorders in human and animal models.^7–9 The underlying mechanisms have been associated with the inhibition of the renin-angiotensin system (RAS) and inflammation in the heart and adipose tissue and in brain regions involved in cardiometabolic regulation including the hypothalamic paraventricular nucleus (PVN).^10–17 There are a few recent studies exploring the effect of exercise on the adverse effects in offspring produced by prenatal challenges.^18–20 In the male offspring of mothers fed a HFD during gestation and lactation, a short period of early postweaning exercise had lasting beneficial effects on body weight, visceral fat, and hormonal profile such as leptin, insulin and triglyceride. However, the effects of exercise on hypothalamic factors controlling metabolism were less marked when compared to offspring of lean dams.^{18, 20} Moreover, maternal HFD reduced offspring responsiveness to the beneficial effects of voluntary exercise to improve endurance capacity, reduce fat mass gain, and ameliorate HFD-induced insulin resistance.¹⁹ These studies focusing on the beneficial effects of exercise on metabolic functions suggest that there is a negative influence of prenatal insults on the capacity of exercise to reduce adverse metabolic effects induced by postnatal HFD. However, there have been no investigations studying whether voluntary exercise can improve cardiovascular dysfunction by reducing BP and increased sympathetic activity produced by prenatal challenges and postnatal HFD.

The PVN is key region in the brain controlling food intake, energy metabolism, sympathetic nervous system (SNS) activity and BP by integrating peripherally and centrally derived humoral and neural information.^{21, 22} Recent studies have described synergistic interactions of the RAS, inflammation and adipokines such as leptin in the PVN as a major mechanism responsible for both human and experimental models of obesity-related hypertension.^23–26 We previously demonstrated that interactions of these three factors enhanced brain reactivity to drive increased SNS activity and the expression of HTRS in HFD fed offspring of mothers with maternal gestational hypertension.^{5, 6, 27} We have also found that voluntary exercise prevented the expression of HTRS elicited by systemic infusion of a slow pressor dose of ANG II.²⁸ Therefore, in the present study our aims were to determine whether voluntary exercise would reverse the sensitized state produced by the maternal hypertension when the expression of HTRS is elicited by postweaning HFD and to investigate the role of central mechanisms. To accomplish this, low-lard-fat diet (LFD) or HFD fed male offspring from normotensive (NT) or hypertensive (HT) dams were given access to running wheel for voluntary exercise. Metabolic markers and changes in BP and autonomic function were determined. Circulating levels of ANG II, proinflammatory cytokine and leptin and putative molecular and cellular mediators in the PVN were also measured. Finally, we assessed the pressor responses to intracerebroventricular (icv) administration of ANG II, tumor necrosis factor (TNF)-α or leptin. The results provide further evidence that voluntary exercise ameliorates the prenatal insult-induced adverse effects exacerbated by a second challenge through a central mechanism. The findings are relevant to gestational hypertension, present-day human lifestyle challenges and postnatal exercise interventions.

Methods

The authors declare that all supporting data are available within the article and in its Data Supplement. The detailed methods are described in the Data Supplement.

Animals

All experiments were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the University of Iowa Animal Care and Use Committee.

All animals were maintained in a temperature (23 ± 2 °C) and light (12-h light/dark cycle) controlled facility. Forty-eight female and forty-eight 10-week-old male rats (Sprague-Dawley, Envigo) were used for breeding. Half of the females were chronically treated with vehicle (saline) throughout mating and pregnancy to serve as NT dams, while the other half were treated with ANG II (subcutaneous, 250 ng/kg/min, model 2004, 4 weeks, Alzet) to produce HT dams. This dose of ANG II infusion induced a significant increase in BP during pregnancy (38.5±6.2 mmHg) in dams.⁴ The offspring were weighed and counted at birth, and the litter sizes reduced at 3 days of age to 4 male and 4 female pups. All offspring were weaned at 3 weeks of age and feeding began with one-half of the animals receiving an LFD (10% calories from lard, 3.85 kcal/g), and the other one-half receiving HFD (60% calories from lard, 5.24 kcal/g) for 12 weeks.

Beginning at 6 weeks of age, all male offspring from both NT and HT dams were given access to either “blocked” (sedentary offspring) or functional running (running/exercise offspring) wheels for 24h/day of 10 weeks during LFD or HFD feeding. Voluntary wheel-running exercise was selected in order to minimize any stressful effects of forced exercise since exogenous or psychological stressors can induce HTRS.^{29, 30} This yielded eight control/experimental groups: 1) Sedentary NT-dam+ LFD-offspring, 2) Running NT-dam+ LFD-offspring, 3) Sedentary NT-dam+ HFD-offspring, 4) Running NT-dam+ HFD-offspring, 5) Sedentary HT-dam+ LFD-offspring, 6) Running HT-dam+ LFD-offspring, 7) Sedentary HT-dam+ HFD-offspring, 8) Running HT-dam+ HFD-offspring. Each experimental group was composed of individual subjects that were randomly selected from different litters. A total of 70 male offspring of NT dams and 72 male offspring of HT dams were used in the present experiments. Food and body weight were weighed one time per week until the experiments ended. Figure S1 shows the experimental methods and timelines for the studies.

Statistical Analyses

Mean arterial pressure (MAP) and heart rate (HR), obtained from the 10 days of telemetry recordings, are presented as daily means and averaged daily values of 10 days of recordings. Differences for BP were calculated for each animal based on the baseline subtracted from the BP after intraperitoneal injection of hexamethonium or icv microinjection of ANG II (5 min), TNF-α (30 min) and leptin (30 min). Likewise, HR differences were calculated for each animal based on the baseline subtracted from the HR after ip injection of atenolol or atropine. HR variability and spontaneous baroreflex sensitivity (SBRS) were obtained by analyzing baseline BP waveforms.^{31, 32} All data were checked for normality and homogeneity of variance by using Shapiro-Wilk test and Levene’s test, respectively. Statistical analyses were performed using two-way repeated measure ANOVA (For the daily MAP and HR and time course of the body weight changes), ordinary two-way ANOVA (For averaged running distances) and three-way ANOVAs (For the rest of the data) (Factors: maternal NT/HT and offspring LFD/HFD or sedentary/exercise). After finding significant interactions between factors, Tukey post-hoc tests were then conducted (Graph-pad Prism 9.0). All data are expressed as means ± SEM. Statistical significance was set at P < 0.05.

Results

Voluntary exercise intensity in LFD or HFD fed offspring of NT and HT dams

The exposure in utero to maternal hypertension and post-weaning HFD feeding had no impacts on exercise intensity in offspring. The 10 weeks of running distances were comparable in all groups of offspring (NT-dam+ LFD-offspring, 8.016±0.997; NT-dam+ HFD-offspring, 8.520±0.917; HT-dam+ LFD-offspring, 8.237±0.854; HT-dam+ HFD-offspring, 8.468±0.695 kilometers/day) (Fig. S2A, B).

Effect of voluntary exercise on metabolic parameters in male offspring

Post-weaning HFD feeding resulted in significant increases in body weight in sedentary offspring of both NT and HT dams when compared to LFD fed sedentary offspring. 10 weeks of exercise significantly reduced the body weight in all groups of offspring, but body weight gain remained higher in HFD fed offspring of both NT and HT dams than that in LFD fed offspring of NT dams (Fig. S3 and Table 1).

Table 1.

Voluntary exercise ameliorated the adverse effects of maternal hypertension and post-weaning high-fat diet (HFD) on metabolism.

Male offspring	n=8-12	NT-LFD	NT-HFD	HT-LFD	HT-HFD
Weaning body weight (g)	sedentary	48.3±1.8	48.9±2.4	46.2±1.6	46.4±2.1
	running	48.3±1.7	50.3±2.2	47.0±3.8	46.4±1.9
Body weight at week 12 (g)	sedentary	385.2±9.2	468.0±12.1ǂ	404.4±8.9	456.1±9.9ǂ
	running	316.4±8.5§	351.5±8.9§	331.4±13.7§	349.5±7.4§
Body weight gain (g)	sedentary	336.9±8.0	419.1±10.0ǂ	358.1±8.1	409.7±7.5ǂ
	running	264.4±5.1§¥	301.2±7.9§	278.4±8.7§¥	303.1±5.4§
Food intake (g/d)	sedentary	16.9±0.4	12.9±0.3ǂ	17.5±0.4	13.1±0.2ǂ
	running	18.5±0.2	13.2±0.2ǂ	18.3±0.2	13.4±0.1ǂ
Energy intake (calories/day)	sedentary	66.4±1.1	67.6±1.5	67.4±1.4	68.4±1.3
	running	71.2±0.9	68.9±1.3	70.5±0.8	70.0±0.4
Energy expenditure (calories/day)	sedentary	47.5±1.4	40.8±1.2ǂ	48.2±1.6	41.9±1.5ǂ
	running	55.6±0.8§	49.2±1.4§¥	54.4±1.1§	50.7±0.7§¥
Feed efficiency (mg BW/calorie)	sedentary	61.5±0.8	73.8±0.2ǂ	63.4±1.1	71.5±1.8ǂ
	running	44.8±1.1§	52.5±1.5§¥	48.0±1.7§	51.6±0.9§¥
Total fat mass (g)	sedentary	23.9±1.8	45.5±2.8ǂ	27.2±1.3	45.0±3.2ǂ
	running	13.0±0.5§	17.1±1.3§	16.0±0.8§	18.3±0.8§
Visceral fat mass (g)	sedentary	10.7±0.6	23.9±1.3ǂ	11.3±0.6	21.4±1.6ǂ
	running	4.0±0.2§	6.2±0.3§	4.1±0.2§	6.4±0.3§
Fat composition (%)	sedentary	6.4±0.4	9.9±0.6ǂ	7.2±0.3	10.1±0.5ǂ
	running	4.1±0.2§	4.6±0.3§	4.9±0.2§	5.0±0.2§
Lean mass (g)	sedentary	212.4±6.6	262.9±7.0ǂ	217.5±6.4	251.6±5.9ǂ
	running	179.8±3.1§	219.3±4.9§¥	186.5±4.9§	204.8±5.1§¥

Changes in the metabolic parameters in sedentary and running male offspring from normotensive (NT) dams or hypertensive (HT) dams after low-lard-fat-diet (LFD) or HFD feeding. (Data are expressed as means ± SEM, n=6/group; Three-way ANOVA was used for analysis followed by Tukey’s post-hoc tests. p<0.05;

^*vs sedentary NT-LFD offspring;

^Ɨvs sedentary NT-LFD offspring;

^#vs sedentary NT-HFD offspring;

^¥vs running NT-LFD offspring;

^§vs corresponding sedentary offspring;

^ǂvs both sedentary and running LFD offspring)

Increased body fat composition was evident in HFD fed sedentary offspring (Fig. S4A), while either total fat mass (Fig. S4B), visceral fat mass (Fig. S4C) or lean mass (Fig. S4D) was significantly increased after 12 weeks of HFD feeding in sedentary offspring from both NT and HT dams when compared with those with LFD. Exercise markedly reduced fat body composition, total fat mass, and visceral fat mass in all groups of offspring and eliminated the differences between LFD and HFD fed offspring. In contrast, exercise significantly decreased lean mass in all groups of offspring, but lean mass of HFD fed offspring remained higher than that in LFD fed offspring (Fig. S4 and Table 1).

Food intake (g/d) was greater in sedentary offspring from both NT and HT dams eating the LFD than those eating the HFD (Fig. S5A). However, caloric intakes (calories/d) were similar among all groups of offspring (Fig. S5B). Energy expenditure (calories/d) was lower in HFD fed offspring when compared with LFD fed offspring (Fig. S5C). As a result, feed efficiency was higher in HFD fed offspring than that in LFD fed offspring (Fig. S5D). Exercise did not alter the pattern of food intake and caloric intake, but significantly increased energy expenditure and decreased feed efficiency in all groups of offspring when compared to the corresponding group of sedentary offspring. However, the energy expenditure remained lower and feed efficiency remained higher in HFD fed offspring than that in LFD fed offspring of NT dams after exercise. (Fig. S5 and Table 1).

Effect of voluntary exercise on increased BP and HR induced by post-weaning HFD in offspring of NT and HT dams

In sedentary offspring, maternal hypertension had no effects on basal MAP (112.3±0.7 vs. 112.5±0.8 mmHg) or HR (334.1±5.4 vs. 329.3±4.6 beats/min). HFD feeding significantly elevated basal MAP in sedentary offspring of both NT (119.9±1.1 mmHg) and HT dams (129.6±0.9 mmHg) when compared to those receiving LFD feeding. However, the increases in MAP were greater in sedentary offspring of HT dams than that in sedentary offspring of NT dams (Fig. 1A,B,C). Exercise did not alter MAP in LFD fed offspring and HFD fed offspring of NT dams (121.3±1.0 mmHg), but significantly reduced MAP in HFD fed offspring of HT dams (120.4±0.7 mmHg), which remained higher than LFD fed offspring (Fig. 1A,B,C).

Figure 1. Voluntary exercise abolished maternal gestational hypertension-induced hypertensive response sensitization to post-weaning high-fat diet (HFD) in adult male offspring.

Changes in mean arterial pressure (MAP, A-C) and heart rate (HR, D-F) in low-lard-fat diet (LFD) or HFD fed male offspring from normotensive (NT) dams and hypertensive (HT) dams after exercise (running). (Data are expressed as means ± SEM, n=6-7/group; Two-way repeated ANOVA or Three-way ANOVA was used for analysis followed by Tukey’s post-hoc tests. p<0.05; # vs sedentary NT-HFD offspring; ǂ vs both sedentary and running LFD offspring; § vs corresponding sedentary offspring).

Dietary treatment had no effect on the HR of any group of offspring. However, exercise significantly reduced the HR in all groups of offspring (Fig. 1D,E,F).

Effect of voluntary exercise on autonomic function, HR variability and baroreflex sensitivity

Ganglionic blockade (Hexamethonium) resulted in a significant reduction in MAP in HFD-fed sedentary offspring when compared to LFD-fed sedentary offspring of either NT dams (Δ−37.1±1.1 vs. Δ−23.4±2.7 mmHg) or HT dams (Δ−50.6±1.0 vs. Δ−25.9±1.9 mmHg). However, the reduction in MAP was greater in sedentary offspring of HT dams than that in sedentary offspring of NT dams. Exercise significantly attenuated the reduction in MAP in HFD-fed offspring of HT dams (Δ−33.9±1.7 mmHg) and did not alter the changes in the HFD-fed offspring of NT dams (Δ−37.3±2.4 mmHg). However, the reduction in MAP in HFD fed offspring of both NT and HT dams remained higher than that in LFD fed offspring, suggesting that voluntary exercise attenuated maternal hypertension-induced HTRS elicited by HFD feeding by reducing sympathetic outflow from the brain, but that it did not normalize the HFD feeding-induced increase in central sympathetic activity (Fig. 2A).

Figure 2. Voluntary exercise ameliorated autonomic dysfunction in male offspring produced by maternal hypertension and high-fat-diet (HFD) feeding.

The autonomic parameters including centrally driven sympathetic activity (A), cardiac sympathetic activity (B) and cardiac vagal activity (C) were obtained by intraperitoneal injection of hexamethonium (Hex), atenolol and atropine, respectively. Heart rate variability [the ratio of low frequency (LF)/high frequency (HF), D] and spontaneous baroreflex sensitivity (SBRS, E) were obtained by spectral analysis of blood pressure waveforms. LFD, low-lard-fat diet; NT, normotensive dams; HT, hypertensive dams. (Data are expressed as means ± SEM, n=6-7/group; Three-way ANOVA was used for analysis followed by Tukey’s post-hoc tests. p<0.05; * vs sedentary NT-LFD offspring; ¥ vs running NT-LFD offspring; # vs sedentary NT-HFD offspring; ǂ vs both sedentary and running LFD offspring; § vs corresponding sedentary offspring).

The β-adrenergic receptor antagonist atenolol significantly decreased HR in HFD-fed sedentary offspring of both NT dams (−66.4±8.0 vs −26.7±6.0 beats/min) and HT dams (−66.9±7.1 vs −21.4±1.8 beats/min). Exercise attenuated these decreases in HR in HFD-fed offspring and eliminated the differences in decreases in HR after β-adrenergic receptor antagonism between LFD and HFD fed offspring, suggesting that exercise blunts HFD-induced increase in cardiac sympathetic activity (Fig. 2B).

HR responses to muscarinic receptor antagonism (atropine) were similar in all groups of sedentary offspring. Exercise significantly increased the HR responses to atropine in all these offspring, indicating that exercise elevates cardiac vagal activity (Fig. 2C).

The spectral analysis indicated that there was an increased low frequency (LF) component attributed to sympathetic modulation (Fig. S6A) and reduced high frequency (HF) component attributed to parasympathetic modulation (Fig. S6B) in HFD fed sedentary offspring of both NT and HT dams. Exercise reversed these LF and HF changes in HFD fed offspring of HT dams, but not in HFD fed offspring of NT dams. As a result, the LF/HF ratio reflecting the sympathetic dominance was significantly increased in HFD fed sedentary offspring of both NT and HT dams, which were reduced by exercise only in HFD fed offspring of HT dams (Fig. 2D). Furthermore, the extent of the reduced LF/HF ratio in HFD fed offspring of HT dams was like those of LFD fed offspring of HT dams and HFD fed offspring of NT dams but remained higher than that in LFD fed offspring of NT dams (Fig. 2D).

SBRS was also markedly reduced in HFD fed sedentary offspring of both NT and HT dams. This depressed baroreflex sensitivity was prevented by exercise (Fig. 2E).

Effect of voluntary exercise on brain reactivity to pressor agents

Either maternal hypertension or HFD feeding alone significantly elevated pressor responses to icv ANG II (Fig. 3A and Fig. S7) and TNF-α (Fig. 3B and Fig. S8) in sedentary offspring. As compared to LFD fed sedentary offspring, HFD feeding significantly elevated pressor responses to icv leptin (Fig. 3C and Fig. S9). Furthermore, the increases in pressor responses to ANG II or leptin were significantly greater in HFD fed sedentary offspring of HT dams than that in HFD fed sedentary offspring of NT dams. Exercise reduced these significant pressor responses to ANG II and leptin and eliminated the differences in pressor responses to either ANG II or leptin between LFD and HFD fed offspring from either NT dams or HT dams. However, exercise did not affect the pressor response to TNF-α in HFD fed offspring of both NT dams and HT dams, suggesting that voluntary exercise did not normalize the enhanced pressor response to TNF-α produced by HFD feeding.

Figure 3. Voluntary exercise modulated brain reactivity to central pressor agents.

The changes in mean arterial pressure (MAP) after intracerebroventricular (icv) injection of angiotensin (ANG) II (A), tumor necrosis factor (TNF)-α (B) or leptin (C) in sedentary and running male offspring from normotensive (NT) dams or hypertensive (HT) dams after low-lard-fat-diet (LFD) or high-fat-diet (HFD) feeding. (Data are expressed as means ± SEM, n=5-6/group; Three-way ANOVA was used for analysis followed by Tukey’s post-hoc tests. p<0.05; * vs sedentary NT-LFD offspring; Ɨ vs sedentary HT-LFD offspring; # vs sedentary NT-HFD offspring; § vs corresponding sedentary offspring)

Effect of HFD feeding and voluntary exercise on plasma ANG II, interleukin (IL)-6 and leptin in offspring

Plasma ANG II was significantly increased in LFD fed sedentary offspring of HT dams and in HFD fed sedentary offspring of both NT and HT dams when compared to LFD fed sedentary offspring of NT dams. Exercise reversed these increases of plasma ANG II (Fig. 4A).

Figure 4. Voluntary exercise reduced the increased plasma pressor agents in male offspring.

Changes in plasma angiotensin (ANG) II (A), IL-6 (B) and leptin (C) in sedentary and running male offspring from normotensive (NT) dams or hypertensive (HT) dams after low-lard-fat-diet (LFD) or high-fat-diet (HFD) feeding. (Data are expressed as means ± SEM, n=7-10/per group; Three-way ANOVA was used for analysis followed by Tukey’s post-hoc tests. p<0.05; * vs sedentary NT-LFD offspring; ¥ vs running NT-LFD offspring; ǂ vs both sedentary and running LFD offspring; § vs corresponding sedentary offspring).

HFD feeding significantly elevated plasma IL-6 in HFD fed sedentary offspring of both NT and HT dams when compared to LFD fed sedentary offspring. Exercise significantly reduced the increased plasma IL-6 in HFD fed offspring, but the levels of IL-6 in HFD fed offspring remained higher than that in LFD fed offspring (Fig. 4B).

HFD feeding significantly elevated plasma leptin in HFD fed sedentary offspring of both NT and HT dams when compared to LFD fed sedentary offspring. Exercise significantly reversed the increased plasma leptin in HFD fed offspring and abolished the differences in levels of leptin between LFD fed offspring and HFD fed offspring (Fig. 4C).

Effect of voluntary exercise on maternal hypertension- and post-weaning HFD-induced mRNA expression of RAS components, proinflammatory cytokines (PICs), leptin and caveolin-1 in the PVN

RT-PCR analysis revealed that HFD feeding resulted in a significant increase in mRNA expression of ANG II type 1 receptor (AT1-R) in the PVN of the sedentary offspring of both NT and HT dams. Exercise had no effect on mRNA expression of AT1-R in LFD fed offspring but reversed upregulation of AT1-R expression in HFD fed offspring (Fig. 5A). Likewise, HFD induced a significant increase in mRNA expression of leptin in sedentary offspring of both NT and HT dams, and the increased expression of leptin was greater in HFD fed offspring of HT dams. Exercise normalized the leptin message expression in HFD fed offspring of both NT and HT dams (Fig. 5B).

Figure 5. Voluntary exercise downregulated mRNA expression of prohypertensive factors in the hypothalamic paraventricular nucleus (PVN).

Comparison of the mRNA expression of renin-angiotensin system components (A), leptin (B), proinflammatory cytokines (C-E) and marker of transcytosis (F) in the PVN of sedentary and running male offspring from both normotensive (NT) and hypertensive (HT) dams after 12 weeks low-lard-fat-diet (LFD) or high-fat-diet (HFD) feeding. (Data are expressed as means ± SEM, n=6/group; Three-way ANOVA was used for analysis followed by Tukey’s post-hoc tests. p<0.05; * vs sedentary NT-LFD offspring; Ɨ vs sedentary HT-LFD offspring; # vs sedentary NT-HFD offspring; § vs corresponding sedentary offspring).

Either maternal hypertension or HFD elicited a significant increase in mRNA expression of TNF-α (Fig. 5C), IL-6 (Fig. 5D) and IL-1β (Fig. 5E) in the PVN of sedentary offspring. Notably, synergy of maternal hypertension and HFD resulted in enhanced increase in the expression of TNF-α and IL-6. Exercise normalized the increased mRNA expression in HFD fed offspring of NT and HT dams and eliminated the differences in message expression of these PICs in all groups of offspring.

Caveoline-1 and claudin-5 are markers of transcytosis and paracellular transport, respectively, that reflect the integrity of the blood-brain barrier (BBB). Increased BBB leakage in the PVN is accompanied by increased expression of caveolin-1 and decreased expression of claudin-5.^{33, 34} Maternal hypertension led to a significant increase in mRNA expression of caveoline-1 in offspring fed with either LFD or HFD. However, exercise resulted in significant decreases in mRNA expression of caveoline-1 and eliminated the differences in caveolin-1 expression in all groups of offspring (Fig. 5F). In contrast, mRNA expression of claudin-5 in the PVN was not altered in any group of offspring (Fig. S10).

Discussion

The goals of the current studies were to determine if voluntary exercise ameliorated experimental maternal hypertension-induced HTRS elicited by postweaning HFD feeding and to investigate the mechanisms mediating any beneficial effect of exercise. The major findings of the present study were that 1) When maintained on LFD, adult offspring of NT dams and HT dams had comparable resting BPs; 2) HFD feeding produced an augmented increase in resting BP (i.e., HTRS) in sedentary offspring of HT dams when compared to sedentary offspring of NT dams.; 3) This HTRS was accompanied by greater sympathetic activity, enhanced pressor responses to centrally administered ANG II or leptin, and enhanced mRNA expression of PICs, leptin and a marker of BBB leakage in the PVN; 4) Exercise significantly reduced the augmented BP and central sympathetic activity in HFD fed offspring of HT dams, but it did not alter the increased BP and central sympathetic activity in HFD fed offspring of NT dams; 5) The plasma IL-6 levels, the pressor response to centrally injected TNF-α, sympathetic activity, and the LF/HF ratio reflecting the sympathetic dominance of HFD fed offspring from both NT and HT dams remained high after exercise. This might account for the higher BP in HFD fed offspring as compared to LFD fed offspring. 6) Nevertheless, exercise had remarkably beneficial effects on metabolic and autonomic function, brain reactivity to ANG II and leptin, and reduced gene expression of brain prohypertensive factors in all offspring; These results indicate that voluntary exercise plays a beneficial role in preventing maternal hypertension-induced HTRS elicited by postweaning HFD and that this is associated with reduction of enhanced brain reactivity and centrally driven sympathetic activity. This study highlights the importance of exercise-related interventions in offspring adversely affected by prenatal factors and/or postnatal aversive environmental challenges, even in offspring from normal mothers.

Obesity-related hypertension in males is mediated primarily through neurogenic mechanisms.²⁵ In obese humans and animal models of diet-induced obesity, increased BP and SNS activity have been demonstrated to be associated with increased brain ANG II, PICs and leptin derived either from de novo synthesis in the brain or from the circulation.^{23, 24, 26} Recent studies showed that prenatal insults exaggerate the cardiometabolic responses of offspring to postnatal adverse factors such as ANG II infusion or HFD.^2–4 In the current and previous studies,⁵ we have implicated several underlying mechanisms contributing to exaggerated centrally initiated sympathetic drive, enhanced pressor responses to centrally acting leptin or prohypertensive RAS components and the sustained increased expression of mRNA for leptin and prohypertensive RAS components in the PVN. These factors are likely to be responsible for HTRS that is expressed under the condition of HFD feeding in adult offspring of HT dams.

We found that there was increased mRNA expression of caveolin-1 in the PVN in offspring of HT dams, but not in offspring of NT dams. Caveoline-1 is a key marker of transcytosis which is an index of the integrity of the BBB. Increased BBB leakage in the PVN, as indicated by the increased expression of caveolin-1, has been shown to be correlated with increased circulating ANG II permeation into the parenchyma, compromised autonomic regulation of sympathovagal balance, augmentation of sympathetic outflow and elevation of BP.^{33, 34} The results of the current study for the first time indicate that maternal hypertension produced increased BBB leakage in offspring. The increased BBB leakage may have facilitated increased access of circulating prohypertensive agents (e.g., ANG II, cytokines, and leptin) produced by HFD feeding to the PVN, a key brain region controlling sympathetic drive and BP. Subsequently, a systemic to central humoral-neural coupling acts to activate brain cardiovascular nuclei leading to a state of HTRS in HT dam offspring.

Exercise of moderate intensity has been proven effective in preventing and managing cardiovascular diseases including heart failure and hypertension.^{35, 36} Central mechanisms including inhibition of RAS activity, reduction of microglial activation, inflammation and oxidative stress, and the restoration of BBB integrity in central cardiovascular nuclei such as the PVN have been demonstrated to result from exercise.^{33, 34, 37, 38} Exercise effectively reduces SNS activity and BP and improves heart failure.^36–38 We previously demonstrated that voluntary exercise normalized the subpressor dose of ANG II-elicited upregulation of mRNA expression of several prohypertensive RAS components, PICs and NADPH oxidase in the brain and that this was accompanied by the abolition of HTRS and attenuation of increased sympathetic activity.²⁸

Although there have been many studies exploring the capacity of interventions (e.g., renal denervation, administration of RAS inhibitors, antioxidants, melatonin, resveratrol, etc.) administered to mothers or their offspring to attenuate or reverse the effects of perinatal developmental programming of cardiometabolic dysfunction,^{4, 39, 40} only a few have investigated the beneficial effects of exercise in offspring with prenatal insults and postnatal overnutrition.^18–20 Recent studies showed that a short period of early postweaning exercise (3 weeks) had lasting beneficial effects on body weight, visceral fat, and hormonal profile in offspring of maternal HFD. However, maternal HFD reduced offspring responsiveness to the beneficial effects of voluntary exercise on the improvement of endurance capacity, reduction of fat mass gain, and amelioration of HFD-induced insulin resistance.^18–20 In this study, we wanted to determine if there was a beneficial effect of long-term voluntary exercise on maternal hypertension-induced HTRS elicited by postweaning HFD feeding. We found that voluntary exercise abolished maternal gestational hypertension-induced elevated sympathetic activity, enhanced pressor responses to centrally administered ANG II or leptin, and greater mRNA expression of PICs, leptin, and especially caveolin-1, a marker of BBB leakage, in the PVN produced by postnatal HFD feeding. As a result, exercise reduced the augmented BP in HFD fed offspring of HT dams to the level seen in HFD fed offspring of NT dams. However, the BP was still higher than that of LFD fed offspring. For the first time, we have demonstrated that exercise blocks the sensitized hypertensive response produced by synergistic interaction of prenatal maternal hypertension and postnatal HFD feeding through increasing BBB integrity, inhibiting central prohypertensive factors and reducing sympathetic outflow. These findings are consistent with recent studies in spontaneously hypertensive rats showing that exercise corrects hypertension-induced BBB dysfunction that is associated with reduction of the brain RAS availability, inhibition of sympathetic outflow and decrease in BP.^{33, 34}

Obesity produced by feeding a HFD is typically characterized by marked elevation of adipokines (leptin), RAS and PICs in the periphery and central nervous system, impairment of autonomic function and baroreflex sensitivity and increased BP.^24–26 Extensive studies have demonstrated that exercise protects against cardiovascular dysfunction produced by obesity/HFD including improvement of baroreflex sensitivity, a reduction of sympathetic activity and attenuations of BP and HR through modifications of prohypertensive RAS and inflammatory responses.^{7–10, 14, 15, 17, 19, 35} Consistent with this, we found that ten weeks of exercise reduced cardiac sympathetic activity and increased cardiac vagal activity and spontaneous baroreflex sensitivity leading to decreases in heart rate, decreased plasma levels of ANG II and leptin and attenuated pressor responses to centrally administered ANG II and leptin. These functional changes were accompanied by reduction of upregulated expression of most prohypertensive genes and a marker of BBB permeability in the PVN in HFD fed offspring of both NT and HT dams. Furthermore, there were no differences in these parameters between LFD fed offspring and HFD fed offspring after exercise, indicating the beneficial roles of exercise in improving HFD feeding-induced cardiovascular dysfunction centrally and peripherally. However, in contrast to other studies showing that exercise reversed the increased BP induced by HFD,^{10, 17} we did not find reduced BP in HFD fed offspring of NT dams, which was comparable to the reduced HTRS in HFD fed offspring of HT dams after exercise.

It is well established that obesity/HFD is closely associated with inflammation in peripheral and central cardiovascular tissues. Various types of exercise have been shown to significantly inhibit inflammatory responses induced by obesity/HFD and to reduce BP.^{8, 17, 41} In the current study, voluntary exercise significantly reduced plasma levels of IL-6 in HFD fed offspring of both NT and HT dams, but it still remained higher when compared to LFD fed offspring. It is possible that even after exercise, high levels of plasma PICs can access the brain to activate key cardiovascular nuclei (e.g., PVN) contributing to enhanced TNF-α elicited pressor action, thereby maintaining increased BP and sympathetic drive observed after ganglionic blockade and elevated sympathetic dominance (LF/HF ratio) in HFD fed offspring of both NT and HT dams. This occurred despite the normalization of upregulated mRNA expression of PICs in the PVN. The discrepancy of the response to PICs and BP changes after exercise between the current study and other reports is not fully understood but may be associated with different HFD composition (low fat 19-30% from peanuts vs. high fat 60% from lard in the current study), the time of initiation of HFD feeding (beginning at adulthood vs. just after weaning in the current study), or different types of exercise (resistance training or forced exercise vs. voluntary exercise in the current study). One or more of these differences might contribute to higher levels of PICs and the maintenance of brain reactivity to these cytokines in HFD fed offspring from both NT and HT dams after exercise in the present study. However, the observation that voluntary exercise normalized plasma levels of RAS components and adipokine leptin and pressor responses to central administration of these agents, but not plasma PIC levels and the pressor responses to a PIC still warrant further investigation.

Excessive HFD intakes lead to producing components of the metabolic syndrome, which includes obesity, elevated RAS activity and inflammation, increased levels of leptin and other metabolic hormones, and insulin resistance. Such metabolic disorders further favor the development of cardiovascular disease.^24–26 Excessive fat accumulation, particularly visceral fat, is associated with increased risk of obesity/HFD-related cardiometabolic diseases that is independent of body mass index.^{9, 11, 42} It has been shown that exercise increased daily energy expenditure, ameliorated visceral adiposity and related metabolic syndrome components by regulating the RAS and the inflammatory response in HFD fed mice.^{11, 13} In the present study, under similar exercise intensity (running distance), voluntary exercise elevated energy expenditure and reduced feed efficiency that led to decreases in body weight gain, total and visceral fat mass and lean mass in all offspring regardless of feeding of LFD or HFD. Although body weight gain, lean mass, feed efficiency remained higher and energy expenditure remained lower in HFD fed offspring when compared with LFD fed offspring, the difference in total and visceral fat mass between LFD fed offspring and HFD fed offspring was eliminated by exercise. This suggests that excess energy resulting from lower expenditure was transferred to lean mass rather than fat mass in HFD fed offspring. Our findings confirm those showing that exercise can produce a greater loss in the abdominal visceral and subcutaneous fat masses even when there is less loss of total body weight,⁴³ and indicate that reduced total/visceral fat was not involved in higher BP levels in HFD fed offspring of both NT and HT dams after exercise.

There are some limitations to the present study. First, besides changes in the neural network that regulates the SNS and BP, the beneficial effects of exercise in obesity-induced cardiovascular disease are also due to a large interplay of cellular and molecular mediators including the RAS, PICs and adipokines in the heart and peripheral vasculature that promotes cardiac remodeling and increases in the endothelium-mediated vasodilatation.^{8, 10–12, 41, 44} We have demonstrated that higher centrally originating sympathetic drive to vessels (i.e. after hexamethonium treatment) and elevated sympathetic dominance (change in LF/HF ratio) were involved in maternal hypertension and post-weaning HFD-induced HTRS. Thus, we cannot rule out the possibility that exercise also affected vascular and heart function directly to attenuate the HTRS in HFD fed offspring of HT dams. Second, in our previous study, we found sex differences in maternal hypertension-induced HTRS to post-weaning HFD. Females from mothers with gestational hypertension also displayed a sensitized hypertensive response to HFD, but it was lower. Due to the interaction between sex hormones and exercise affecting food intake and energy balance⁴⁵ and different mechanisms underlying the sensitizing effect of maternal hypertension on dietary treated male and female offspring,^{5, 6} we will explore beneficial effects of voluntary exercise on maternal hypertension-induced HTRS to post-weaning HFD in female offspring in separate studies.

Perspectives.

This study demonstrated that voluntary exercise abolishes maternal gestational hypertension-induced HTRS to the post-weaning HFD in adult male offspring, which is associated with attenuation of enhanced brain reactivity and central sympathetic activity by centrally inhibiting the effects of brain RAS and leptin and by increasing BBB integrity. This study provides insight into the central mechanisms underlying exercise improvement of the cardiometabolic dysfunction synergistically produced by prenatal insults and postnatal overnutrition. This study also highlights the importance of early exercise-related lifestyle intervention as a non-pharmacological therapy for prevention of obesity and hypertension. However, more studies on how exercise regulates systemic to central humoral-neural coupling and the circuit within the hypothalamic nuclei involved in processing signals associated with the prohypertensive factors that protects against the maternal hypertension and HFD-induced sensitization should be specifically studied in the future.

Novelty and Relevance.

What Is New?

• These studies demonstrate that voluntary exercise abolishes maternal gestational hypertension-induced hypertensive response sensitization elicited by the post-weaning high-fat diet in adult male offspring. This beneficial effect is associated with reduction of enhanced brain reactivity and central sympathetic activity through centrally inhibiting the effects of the brain renin-angiotensin system and leptin and increasing blood-brain barrier integrity.

What Is Relevant?

• The data indicate that exercise improves the cardiometabolic dysfunction synergistically produced by prenatal insults and postnatal overnutrition, which are relevant to maternal hypertension, present-day human lifestyle challenges and exercise interventions.

Clinical/Pathophysiological Implications

• These findings help us understand the central mechanisms underlying exercise improvement of sensitized obesity-related hypertension, and early exercise-related lifestyle intervention reducing brain responsiveness to prohypertensive agents and sympathetic activity is effectively non-pharmacological therapy for prevention of obesity and hypertension.

Nonstandard Abbreviations and Acronyms

ANG II

angiotensin II

AT1-R

angiotensin II type 1 receptor

BBB

blood-brain barrier

BP

blood pressure

HFD

high-fat diet

HR

heart rate

HT

hypertensive

HTRS

hypertensive response sensitization

icv

intracerebroventricular

IL

interleukin

MAP

mean arterial pressure

LFD

low-lard-fat diet

NT

normotensive

PICs

proinflammatory cytokines

PVN

hypothalamic paraventricular nucleus

RAS

renin-angiotensin system

SNS

sympathetic nervous system

SBRS

spontaneous baroreflex sensitivity

TNF

tumor necrosis factor