Disclosure: The authors declare no conflict of interest.
Funding agencies: This work was supported by grants from the Associacão Fundo de Incentivo à Pesquisa (AFIP) and the Fundação de Amparo à Pesquisa do Estado de São Paulo (CEPID #98/14303-3). ST, MLA, CTB and RRC are recipients of fellowships from CNPq.
There is a reciprocal relationship between sleep duration and weight gain. However, the consequences of this relationship on the cardiovascular system over an entire life span are still not fully elucidated. We examined the effect of acute sleep deprivation (SD) on baroreflex sensitivity and blood pressure in Zucker rats of different ages.
Design and Methods:
Female lean and obese Zucker rats at 3, 6 and 15 months of age were assigned to SD or control (CTRL) groups. After a 6 h period of the SD procedure (6 h of gentle handling) or CTRL procedure (an equivalent period without handling), the animals were anesthetized for surgical catheterization of the femoral artery and vein. To evaluate the baroreflex sensitivity index, bolus infusions of phenylephrine (bradycardia response) and sodium nitroprusside (tachycardia response) were administered.
Obesity resulted in dysfunctional tachycardia responses at 3 months of age. At 6 and 15 months of age, both bradycardia and tachycardia responses were significantly lower in obese animals than those in lean animals. At 15 months of age, interactions among obesity, SD and aging produced the most marked effects on the cardiovascular system (increased mean arterial pressure and heart rate and decreased baroreflex sensitivity).
Therefore, these results suggest that there is no direct relationship between baroreflex imbalance and an increase in arterial pressure.
Modern living imposes stress factors that can individually and collectively curtail optimal sleep. In addition, studies have shown a close relationship between sleep deprivation (SD) and obesity (1, 2). For example, in a chronic study, Patel and colleagues (3) reported that in a study of approximately 70,000 women, those who slept 5 h or less weighed ∼2.47 kg more than those who slept 7 h. Furthermore, obese individuals usually have difficulty getting a good night's sleep. Weight gain can have important negative health effects, especially upon the cardiovascular system (4, 5). Moreover, another important risk factor for cardiovascular disease is sleep loss. Although a significant body of literature describes the effect of chronic sleep loss, little attention has been paid to acute sleep loss, and therefore, its repercussions on health and well-being are still unknown.
The association between sleep loss and cardiovascular disease may be due to effects of altered autonomic nervous system activity on cardiovascular physiology (6). Some hypotheses suggest that vascular sympathetic activation is involved in increased blood pressure after SD, in part due to abnormalities of the baroreflex control response (6, 7). The cardiac baroreflex plays a critical role in the acute regulation of sympathetic vasomotor activity and arterial pressure, but its importance in the long-term control of these parameters is still controversial (8). Previous findings indicate that the baroreflex chronically influences the degree of sympathetic activity and that baroreflex dysfunction could significantly contribute to chronic sympathetic activation and the resulting hypertension (9).
Notably, women generally have a greater fat mass than men and appear to be more affected by SD (10). Although the incidence of cardiovascular events is increasing in women, men also have an increased risk.
Moreover, obesity is associated with changes in the autonomic regulation of arterial pressure and vascular reactivity to sympathetic stimulation (11).
The effects of obesity and SD are typically investigated independently. Accordingly, the association between these factors has not been thoroughly explored, although such studies may provide information relevant to health and disease throughout the life span. Normal aging is a universal phenomenon that is accompanied by a complex series of changes in autonomic control of the cardiovascular system. These changes favor heightened cardiac sympathetic tone with parasympathetic withdrawal due to blunting of cardiovagal baroreflex sensitivity (12). The obese Zucker rat demonstrates cardiovascular dysfunction with increasing age (13); however, previous studies have only used rats of ∼2 months old. Although the effect of aging on the sympathetic nervous system has been investigated, conflicting data have been reported in Zucker rats.
Genetically obese rodent models have been widely used to understand the etiology of obesity. Although obese animals cannot serve as exact models of the human condition, they have provided important insights into the causes of human obesity and its possible consequences. The obese phenotype in Zucker-fatty rats is the result of a spontaneous mutation. The obesity is transmitted as an autosomal Mendelian recessive trait, and homozygous animals are hyperphagic and experience the consequences of genetic obesity. Obese Zucker rats accumulate fat progressively throughout their lives and show certain abnormalities, such as hyperinsulinemia (14) insulin resistance, hyperlipidemia (15), increased efficiency for food use and decreased physical activity (16).
Therefore, the purpose of this study was to investigate the effect of single and multiple cardiovascular risk factors (SD, obesity and aging) on baroreflex sensitivity and cardiovascular parameters in lean and obese female Zucker rats. Our results could be used to identify factors associated with baroreflex dysfunction and hypertension in this animal model of obesity.
Methods and Procedures
Experiments were performed on female lean and obese Zucker rats provided by the Centro de Desenvolvimento de Modelos Experimentais para Medicina e Biologia (CEDEME) of Universidade Federal de São Paulo. The animals were housed in groups of three in standard polypropylene cages, maintained at 22°C with a 12:12 h light-dark cycle (lights on at 7 am), and allowed free access to food and water. The rats were maintained and treated in accordance with the guidelines established by the Ethical and Practical Principles for the Use of Laboratory Animals (17). All animal procedures were approved by the University Ethics Committee (Protocol #1268/08).
Acute sleep deprivation
The animals in the present study were subjected to a single episode of continuous SD during the first 6 h of the light photoperiod, which is the typical sleep period for rats (7 am to 1 pm). The SD was carried out by a gentle handling method, introducing an object (a pick) into the cage and tapping the cages whenever the animals appeared drowsy. The animals were not disturbed during feeding and drinking (18). Additionally, the platform technique for SD was not the preferred method because obese rats would have difficulty standing on a narrow platform as required by the platform technique protocol.
Female lean and obese Zucker rats at 3, 6, and 15 months of age were randomly assigned to either the control (CTRL) or SD protocol (n = 8 to 9 rats per group). Experiments were started in all rats in the diestrus phase of the estrous cycle because we previously demonstrated that this phase is particularly affected by SD (19).
Rats assigned to the SD-group were handled gently for 6 h, whereas CTRL rats were allowed to sleep. After the single SD period, all animals were subjected to surgical vascular catheterization. The rats were initially anesthetized by halothane inhalation (5%) and were maintained on a mix of 3% halothane with 100% O2. Tapered polyethylene catheters (PE-50) were placed in the right femoral artery to monitor arterial pressure and in the right femoral vein to infuse drugs (sodium nitroprusside and phenylephrine). It is widely accepted that halothane is a lipo-soluble anesthetic, and exposure must be prolonged, even for several hours, before those effects can be observed. In our study, it took 1 min to induce anesthesia and 10 min to maintain it in our experimental animals, the length of time our experiment consumed. The procedure was always carried out by the same trained professional. In this setting, there was insufficient time for the formation of halothane deposits in adipose tissue. The induction and recovery from anesthesia occurred quickly and superficially by means of the upper airways.
After the animals recovered normal physiological function (minimum of 30 min after catheterization), baseline values of mean arterial pressure and heart rate were recorded in conscious and freely moving rats. The arterial pressure was measured using a Powerlab Data Acquisition System (PowerLab/8SP; ADInstruments, Colorado Springs, CO) connected to a pressure transducer (BP-100RE; CB Sciences, Dover, NH) at a sampling rate of 2 kHz. Each animal then received three bolus injections (0.1 mL) of phenylephrine (30, 50, and 100 μg mL−1) and sodium nitroprusside (50, 150, and 200 μg mL−1) in random order.
The changes in the peak value of the mean arterial pressure and the reflex changes in heart rate in response to injections of pressor (phenylephrine-bradycardia response) and depressor (sodium nitroprusside-tachycardia response) agents were used to quantify baroreflex sensitivity (beats/mmHg) as the ΔHR/ΔMAP ratio for each animal.
Initially, homogeneity of variance was tested using Levene's test and normality of data distribution with the Kolmogorov-Smirnov test in all analyses. Whenever these tests showed absence of homogeneity and normality, data were analyzed using Z-score transformation. The Student's t test was used to compare body weights of lean and obese rats at each age. Two-way analysis of variance (ANOVA) followed by Duncan's post hoc test was used to independently compare groups (CTRL and SD) and conditions (lean and obese) as a function of age (3, 6, and 15 months of age). These analyses were used to verify differences in the mean arterial pressure, heart rate and baroreflex gain (bradycardia and tachycardia responses). Data are presented in the figures and text as means ± SD.
Body weight in lean and obese CTRL and SD female rats at 3, 6, and 15 months of age
As expected, at 3 months old, adult obese rats showed a significant increase in body weight compared with that in the lean group (404.0 ± 35.8 vs. 205.3 ± 16.2 g; P ≤ 0.001). However, SD did not alter body weight in either lean or obese rats. Similar to younger rats, SD had no effect on the weight of adult obese rats, which was significantly higher than that of the lean rats (544.3 ± 56.6 vs. 241.4 ± 23.4 g; P ≤ 0.001). Similar to the younger rats, there was a statistically significant increase in weight of adult obese rats compared with that in lean rats (747.0 ± 42.3 vs. 259.2 ± 45.6 g; P ≤ 0.001). However, SD did not significantly affect body weight.
Mean arterial pressure in lean and obese CTRL and SD female rats at 3, 6, and 15 months of age
In relation to general statistics for cardiovascular parameters, two-way ANOVA showed that, at 3 months old, there was a significant effect on condition (lean × obese, P ≤ 0.01) and group (control × sleep deprivation, P ≤ 0.001) and a significant interaction between the two factors (P ≤ 0.001). Two-way ANOVA showed that, at 6 months old, there was a significant effect of condition (lean × obese, P ≤ 0.0001) and group (control × sleep deprivation, P ≤ 0.01) and a significant interaction between the two factors (P ≤ 0.01). Two-way ANOVA showed that, at 15 months old, there was a significant effect of condition (lean × obese, P ≤ 0.0001) and group (control × sleep deprivation, P ≤ 0.0001), and there was a significant interaction between the two factors (P ≤ 0.03).
At 3 months old, mean arterial pressure was significantly higher in obese-CTRL than that in lean-CTRL rats (112.8 ± 4.8 vs. 101.1 ± 9.8 mmHg; P ≤ 0.01, Table 1) and it was significantly higher in the lean-SD group compared with that in the lean-CTRL group (111.7 ± 6.5 vs. 101.1 ± 9.8 mmHg; P ≤ 0.01, Table 1). At 6 months old, mean arterial pressure was significantly higher in the obese-CTRL group than that in the lean-CTRL group (116.0 ± 6.8 vs. 103.9 ± 5.4 mmHg; P ≤ 0.001, Table 1). In obese-SD rats mean arterial pressure was significantly lower than that in obese-CTRL rats (110.1 ± 1.5 vs. 116.0 ± 6.8 mmHg; P ≤ 0.05, Table 1). At 15 months old, mean arterial pressure was significantly higher in lean-SD rats compared with that in lean-CTRL rats (111.7 ± 6.5 vs. 100.0 ± 11.7 mmHg; P ≤ 0.01, Table 1). Similarly, obese-SD rats showed an increase in mean arterial pressure compared with obese-CTRL rats (113.9 ± 4.9 vs. 101.3 ± 10.8 mmHg; P ≤ 0.01, Table 1).
Table 1. Mean arterial pressure (MAP) and heart rate (HR) in control (CTRL) and sleep deprived (SD) lean and obese Zucker rats at 3, 6, and 15 months of age
Shown are the means ± SD (n = 8/9 rats/group).
Different from age-matched CTRL.
Different from age-matched lean. P ≤ 0.05 (ANOVA two-way, Duncan test).
Heart rate in lean and obese CTRL and SD female rats at 3, 6, and 15 months of age
No significant changes in heart rate were found in the groups tested at 3 months old. At 6 months old, heart rate was significantly lower in the lean-SD group than that in the lean-CTRL group (375.8 ± 14.6 vs. 418.0 ± 17.8 bpm; P ≤ 0.01, Table 1). SD caused a significantly higher heart rate in the obese group than that in the lean-SD group (413.8 ± 19.4 vs. 375.8 ± 14.6 bpm; P ≤ 0.01, Table 1). At 15 months old, heart rate was higher in the lean-CTRL group compared with that in the obese-CTRL group (400.3 ± 7.2 vs. 361.6 ± 49.3 bpm; P ≤ 0.05, Table 1), and the obese-SD group also showed a significantly higher heart rate than that in the obese-CTRL group (422.9 ± 24.6 vs. 361.6 ± 49.3 bpm; P ≤ 0.001, Table 1).
Bradycardia response in lean and obese CTRL and SD female rats at 3, 6, and 15 months of age
Bradycardia evoked by baroreflex activation as a result of phenylephrine-induced arterial pressure rise was attenuated in the obese-SD group compared with the corresponding CTRL group (−1.1 ± 0.4 vs. −2.5 ± 0.3 beats/mmHg; P ≤ 0.001, Figure 1A). Moreover, the combined effects of SD and obesity resulted in a significant baroreflex imbalance in obese-SD rats compared with lean-SD rats (−1.1 ± 0.4 vs. −2.7 ± 0.8 beats/mm Hg; P ≤ 0.001, Figure 1A). At 6-months old, the bradycardia reflex caused by the phenylephrine-induced arterial pressure rise was attenuated in obese-CTRL rats in relation to lean-CTRL rats (−1.3 ± 0.6 vs. −2.1 ± 0.5 beats/mm Hg; P ≤ 0.01, Figure 2A). Similarly, obesity combined with SD caused baroreflex imbalance above that of lean-SD rats (−1.7 ± 0.3 vs. −2.4 ± 0.1 beats/mm Hg; P ≤ 0.01, Figure 2A). As depicted in Figure 3A, the baroreflex sensitivity, as assessed by the bradycardic response to arterial pressure rise, was significantly decreased in the obese-CTRL group compared with that in the lean-CTRL group (−0.8 ± 0.3 vs. −2.2 ± 0.3 beats/mm Hg; P ≤ 0.001, Figure 3A) and it was significantly decreased in the obese-SD group compared with that in the lean-SD group (2.7 ± 0.9 vs. −1.8 ± 0.3 beats/mmHg; P ≤ 0.001, Figure 3A).
Tachycardia response in lean and obese CTRL and SD female rats at 3, 6, and 15 months of age
Reflex tachycardia provoked by arterial hypotension in response to injections of sodium nitroprusside was affected by obesity, age and SD. At 3 months old, SD significantly decreased sympathetic baroreflex sensitivity in obese-SD rats compared with that in CTRL rats (−0.6 ± 0.3 vs. −1.8 ± 0.7 beats/mmHg; P ≤ 0.001, Figure 1B). Sympathetic baroreflex sensitivity was also significantly decreased in the obese-SD group compared with that in the lean-SD group (−0.6 ± 0.3 vs. −1.4 ± 0.5; P ≤ 0.01, Figure 1B). At 6 months old, the baroreflex gain was also attenuated in the obese-CTRL group compared with that in the lean-CTRL group (−1.3 ± 0.4 vs. −3.3 ± 0.5 beats/mmHg; P ≤ 0.01, Figure 2B), and it was decreased in the obese-SD group compared with that in the lean-SD group (−1.2 ± 0.5 vs. −2.8 ± 0.1 beats/mmHg; P ≤ 0.01). Sympathetic baroreflex sensitivity was also significantly decreased in the lean-SD group compared with that in the lean-CTRL group (−2.8 ± 0.1 vs. −3.3 ± 0.5 beats/mmHg; P ≤ 0.05, Figure 2B). At 15 months old, analysis of the reflex tachycardia caused by sodium nitroprusside showed a marked effect of obesity on baroreflex response, which was lower in obese-CTRL rats than that in lean-CTRL rats (−0.8 ± 0.1 vs. −2.1 ± 0.4 beats/mmHg; P ≤ 0.001, Figure 3B). A significantly lower baroreflex sensitivity was observed in obese-SD rats than that in obese-CTRL rats (−0.6 ± 0.3 vs. −0.8 ± 0.1 beats/mmHg; P ≤ 0.001, Figure 3B). In addition, SD significantly reduced the baroreflex response in lean-SD rats compared with lean-CTRL rats (−1.4 ± 0.5 vs. −2.1 ± 0.4 beats/mmHg; P ≤ 0.001, Figure 3B) and in obese-SD animals compared with lean-SD animals (−0.6 ± 0.3 vs. −1.4 ± 0.1 beats/mmHg; P ≤ 0.001, Figure 3B).
Among the risk factors analyzed (SD, obesity and aging), obesity exerted the greatest effect on cardiovascular autonomic imbalance. The sympathetic dysfunction began at 3 months of age in obese rats. When the risk factors were analyzed independently, obesity was found to exert a greater effect on cardiovascular parameters than SD at 6 and at 15 months of age. However, the interaction between obesity and detrimental effects of SD were more severe in the oldest group of animals. In relation to mean arterial pressure, the effect of risk factors depended on age. Therefore, at an age of 3 and 6 months, obesity caused elevated mean arterial pressure in CTRL rats. At 15 months of age, SD elevated mean arterial pressure in obese and in lean rats.
It has been reported that increased body weight is associated with changes in autonomic control of the cardiovascular system (20). Obese individuals suffer from an increased mortality risk, arguably due to cardiovascular disorders stemming from reduced parasympathetic or increased sympathetic activity (21). Hirsch et al. (22) described an inverse relationship between the extent of weight gain and the resulting decrease in parasympathetic control. The protein hormone leptin could be associated with this effect, as obese Zucker rats have high plasma leptin concentrations (23, 24). Leptin alters satiety and contributes to hyperphagia and weight gain. Leptin is secreted by fat cells, crosses the blood-brain barrier (25) and activates neurons located in several hypothalamic areas, including the paraventricular nucleus (26, 27).
In addition to its role in the maintenance of energy balance, leptin also participates in the central regulation of cardiovascular functions (28). Central application of leptin augments sympathetic outflow resulting in an increase in systemic arterial pressure (28), which appears to be mediated by interaction with the arterial baroreflex system (29). Furthermore, the neuroendocrine regulation of appetite and food intake appears to be influenced by sleep duration, and sleep restriction may favor the development of obesity because sleep plays an important role in energy balance (30). A study by Laposky et al. (30) indicated that impaired leptin signaling has deleterious effects on the regulation of sleep amount, sleep architecture, and temporal consolidation of arousal states. The authors concluded that leptin may represent an important molecular component in the integration of sleep, circadian rhythms, and energy metabolism. Indeed, sleep is an important modulator of neuroendocrine function and glucose metabolism, and sleep loss has been shown to result in metabolic and endocrine alterations, including decreased glucose tolerance, insulin sensitivity and leptin, as well as increased levels of ghrelin, hunger and appetite (31). It has been reported that feelings of hunger as well as plasma ghrelin levels were already elevated after one night of sleep deprivation, whereas morning serum leptin concentrations remained unaffected (31). This association is particularly interesting in obese Zucker rats that have high plasma leptin concentrations. Concomitant monitoring of energy expenditure and peripheral leptin and ghrelin concentrations enabled us to assess the role of several factors that have been hypothesized to be linked with a lack of sleep and increased energy intake (31). Epidemiological studies showed a distinct relationship between sleep duration and body weight (32, 33). Collectively, these data suggest a relationship between these two factors in that both shortened and extended sleep durations coincide with increases in body weight, a reduction in circulating leptin concentrations known to suppress appetite (34), and elevated ghrelin concentrations, which promote hunger (33). Therefore, these findings indicate a distinct influence of sleep and sleep loss on endocrine regulation of energy homeostasis. In fact, sleep plays an important role in energy balance (34). A recent epidemiological study indicated a close relationship between sleep duration and body weight (35). Cardiovascular disturbances have been suggested to affect endothelial function during both short and prolonged SD (36). It is well known that obesity is characterized by sympathetic nervous system activation, and there is considerable evidence that this system plays an important role in the initiation and maintenance of hypertension (37).
In the current study, baroreflex dysfunction beginning at 3 months of age in obese rats led to an increase in mean arterial pressure at 15 months of age. However, the obese-CTRL group did not show an increase in mean arterial pressure at 15 months of age despite clear evidence of baroreflex imbalance, which was already in an early stage at 6 months of age. In contrast, SD at 15 months of age significantly increased mean arterial pressure in lean animals; however, baroreflex dysfunction was evident only by altered reflex tachycardia. Taken together, these data suggest that there is no direct relationship between baroreflex imbalance and increased arterial pressure. The hyporesponsive cardiac baroreflex itself is a negative prognostic factor for cardiovascular disease and an important marker of low survival regardless of baseline hemodynamic findings (38).
Several studies have indicated that baroreflex resetting is incomplete in hypertension (9, 39) and that baroreflex suppression of sympathetic activity is a sustained response during hypertension that may serve as a compensatory mechanism to lower blood pressure (9). Current dogma suggests that the baroreflex is relatively unimportant in long-term control of arterial pressure (39).
In the present study, we measured blood pressure at a minimum of 30 min after surgical implantation of femoral artery catheters, and it is possible that baroreflex imbalance in the obese rats was a consequence of a disproportionate reaction to the surgical stress involved. However, we observed no evidence of postoperative pain or suffering in either the lean or obese rats. Indeed, the duration of surgery and of recovery from anesthesia was similar in both groups. The overall appearance and behavior of the obese and lean rats were also similar.
Notably, we demonstrated that the risk factors of obesity, SD and aging were detrimental to the cardiovascular system, and of the three factors, obesity caused the most damage. In the young animals, acute SD alone did not alter any of the cardiovascular parameters examined, but aging subjects readily demonstrated additional cardiovascular risk attributable to compensatory reflexes typically observed in older animals. Moreover, the present study supports the hypothesis that baroreflex dysfunction does not contribute directly to chronic sympathetic activation during hypertension because baroreflex function is mainly involved in short-term neural regulation of blood pressure.
This study evaluated the acute effect of SD. However, the association between different sleep durations and the risk of all-cause death and cardiovascular events needs to be clarified. Analysis of the cardiac baroreflex is an important predictor of cardiovascular morbidity; however, it is a peripheral response, and therefore, other measurements such as sympathetic nervous activity or brain functions related to blood pressure control might provide further accurate mechanisms. Our study addressed the consequences of short term sleep deprivation and obesity across the life span of Zucker rats. The current findings provide evidence of the effect of only one aspect of sleep deprivation in the cardiovascular system.
This work was supported by grants from Associação Fundo de Incentivo à Pesquisa (AFIP) and Fundação de Amparo à Pesquisa do Estado de São Paulo (#11/12325-6 to T.A.A., #10/50129-1 to C.H.). M.L.A., C.T.B., and S.T. are recipients of CNPq fellowships.
The authors gratefully acknowledge the valuable assistance of Marilde Costa and Waldemarks Leite. The authors declare that there are no conflicts of interest.