INTRODUCTION

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Keywords: estradiol; leptin; sleep; ventilation; obesity

Obesity and male gender are both clinical risk factors for the development of sleep disordered breathing (1). Obesity is often associated with a depression of respiratory control mechanisms, particularly during sleep, leading to a decrease in neural input to the upper airway (obstructive sleep apnea) or the diaphragm (obesity hypoventilation syndrome) (2). Sex can also affect respiratory control, and females are known to have less severe sleep-disordered breathing than males at comparable degrees of obesity (1, 9). Although such sex differences have been attributed to the respiratory effects of sex steroids (7, 10), little is known about the influence of sex on respiratory control in obese individuals.

Obesity itself is now thought to influence respiratory control. We have recently shown in a mouse model of obesity hypoventilation that leptin, a neurohumoral factor inhibiting food intake (14), is a powerful modulator of respiratory control (15, 16). The absence of leptin in the C57BL/6J-Lep^ob mouse induces profound obesity, respiratory depression, and elevated Pa_CO2. Furthermore, the effects of leptin deficiency on respiratory depression, and the effects of leptin administration on augmenting respiratory control, were more pronounced during sleep than wakefulness (16). Thus, increases in leptin help compensate for adiposity by maintaining respiratory control during wakefulness and sleep. Although leptin is positively correlated with body mass index (BMI) (17, 18), its levels are known to be at least double in females compared with males of equivalent BMI (17, 19). Nevertheless, it is unknown whether leptin influences respiratory control differently in obese females compared with obese males.

Therefore, the purpose of the present study was to examine the relationship between leptin and sex on respiratory control in normal and obese mice during wakefulness and sleep. We hypothesized that the elevated leptin levels in females may be necessary to maintain normal respiratory control in the presence of obesity, and that leptin deficiency in females might lead to particularly severe respiratory depression. The results from our study indicate that leptin is even more important in females than males in preventing the previously described respiratory depression associated with obesity.

	METHODS

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Animals

Seventeen mutant, obese C57BL/6J-Lep^ob mice (8 female and 9 male) and 32 wild-type C57BL/6J mice (16 female and 16 male) from Jackson Laboratory (Bar Harbor, ME) were used in the study. The study was approved by the Johns Hopkins University Animal Use and Care Committee and complied with the American Physiological Society Guidelines. For all surgical procedures, anesthesia was induced and maintained using halothane administered through a face mask. At the completion of experiments, animals were euthanized with pentobarbital (60 mg intraperitoneally).

Ventilatory Measurements during Sleep/Wake

Mice were instrumented with chronically implanted polysomnographic electrodes for determination of sleep/wake state as previously described (16). During data collection periods, the animals were placed in a whole body barometric plethysmography chamber to measure ventilation as previously described (15, 16). Ventilation was measured during wakefulness, non-rapid eye movement (NREM) and REM sleep in response to a range of hypercapnic gases (0, 3, 5 and 8% CO₂ in 40% O₂ to ensure no hypoxic stimulus). The animals were allowed to freely cycle through sleep/wake states. Tidal volume and respiratory frequency were assessed over 5- to 8-s periods during wakefulness and NREM sleep. In REM sleep, tidal volume and frequency were highly variable. Thus, ventilatory measurements were made over the entire period of REM sleep, which had to be 30 s or longer for inclusion. The hypercapnic ventilatory response (HCVR) was determined in each animal by the slope of the relationship between minute ventilation ( E) and inspired CO₂ (0-8%) during wakefulness and NREM sleep via linear least-squares regression analysis. During REM sleep the HCVR was calculated over 0-5% because the animals could not consistently maintain 30-s periods of REM sleep during 8% CO₂ challenge.

Experimental Groups

Comparisons of ventilatory control parameters were made between weight-matched female and male mice in three separate groups: (1) obese C57BL/6J-Lep^ob mice maintained on a regular ad libitum chow diet, (2) wild-type mice maintained on a regular ad libitum chow diet, and (3) wild-type mice maintained on a high fat diet (49% fat; 5.8 kcal/g) for approximately 16 wk as previously described (16).

Radioimmunoassay

Arterial blood (0.8-1.2 ml) was obtained from direct cardiac puncture under halothane anesthesia. After blood withdrawal the animals were euthanized with pentobarbital (60 mg intraperitoneally). Serum leptin levels were measured with a mouse leptin radioimmunoassay kit from Linco Research, Inc. (St. Charles, MO). The intraassay coefficient of variation was 7.2%. Serum estradiol and progesterone levels were measured with radioimmunoassay kits from Diagnostic Products Corp. (Los Angeles, CA).

Statistical Analyses

Data were analyzed using Crunch 4 (Crunch Software Corporation; Oakland, CA), and results presented for E and HCVR are shown as mean ± SEM. Significance between sex or degree of obesity was derived within each sleep/wake state using one-way, within-subject ANOVA with Newman-Keuls post hoc analyses where appropriate.

	RESULTS

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The age, weight, and hormonal status for female and male mice are shown for each of the three experimental groups in Table 1. Leptin levels were higher for both the regular diet (p < 0.005) and high fat diet (p < 0.025) wild-type females compared with wild-type males in respective group.

Our approach of weight-matching male and female mice led to age discrepancies between some groups (Table 1). Wild-type male mice (unlike C57BL/6J-Lep^ob mice) gained weight at a faster rate than females, and hence were younger under conditions of weight matching. However, all mice were between 75 and 225 d old, an age range over which the HCVR reaches a stable plateau in both wild-type and C57BL/6J- Lep^ob mice (15).

Ventilatory Responses to Hypercapnia

C57BL/6J-Lepob. The ventilatory response to CO₂ challenge during wakefulness and sleep is shown for male and female C57BL/6J-Lep^ob mice in Figure 1. Baseline ventilation (0% CO₂; left panel) was comparable for males and females across each of the sleep/wake states. The HCVR, however, was significantly reduced in female compared with male mice during both wakefulness and NREM sleep (Figure 1, right panel). During NREM sleep the depressed HCVR of 3.1 ± 0.5 ml/ min/% CO₂ observed in male mice was further halved (p < 0.03) to 1.5 ± 0.5 ml/min/% CO₂ in female mice. In REM sleep neither the male nor female mice had HCVR values significantly different from zero.

View larger version (20K): [in this window] [in a new window]   Figure 1.   Minute ventilation ( E, left panel  ) at increasing levels of inspired CO2 and the hypercapnic ventilatory response (HCVR, right panel  ) across sleep/wake states are compared between male and female C57BL/6J-Lepob mice. Results presented show the mean ± SEM. Significance between sexes was derived within each sleep/wake state using one-way, within-subject ANOVA.

The respiratory frequency was lower in females compared with male C57BL/6J-Lep^ob mice, but the change in respiratory frequency in response to CO₂ challenge was not affected by sex during wakefulness or NREM sleep (Table 2). In contrast, the female mice demonstrated a significantly reduced respiratory drive in response to CO₂ challenge compared with male mice during wakefulness and NREM sleep (Table 3), as determined by VT/TI (ml/s). For example, during NREM sleep the 8% CO₂ challenge caused an increase in VT/TI of 0.35 ± 0.16 ml/s in female mice compared with an increase of 0.92 ± 0.14 ml/s in male mice for the same stimulus (p < 0.025). In REM sleep, the baseline VT/TI was identical between female and male mice and there was no increase during CO₂ challenge.

No significant differences in either estradiol or progesterone levels were noted between males and females. In female C57BL/6J-Lep^ob mice serum estradiol level was 4.0 ± 1.3 ng/ ml and progesterone was 3.8 ± 0.6 ng/ml compared with 4.5 ± 2.0 ng/ml and 5.5 ± 0.3 ng/ml, respectively, in male C57BL/6J- Lep^ob mice.

Regular diet C57BL/6J. We next tested whether female wild-type mice were capable of normal ventilatory responses to CO₂ challenge. Figure 2 is a sample trace showing that the baseline minute ventilation during wakefulness was comparable between a female wild-type and a female C57BL/6J-Lep^ob mouse, despite the obese mouse weighing almost three times that of the wild-type mouse. During 8% CO₂ challenge, however, the wild-type mouse had a minute ventilation that was double that of the obese mouse (Figure 2).

View larger version (33K): [in this window] [in a new window]   Figure 2.   Representative samples of EEG, EMG, and tidal volume (VT) fluctuations during wakefulness in one female C57BL/6J mouse (upper) and in an obese C57BL/6J-Lepob mouse (lower) at baseline (left panels) and during 8% CO2 challenge (right panels).

Pooled data for ventilatory responses to CO₂ challenge in regular diet wild-type mice and the C57BL/6J-Lep^ob mice are shown in Figure 3. The HCVR for the regular diet mice was approximately three times greater during wakefulness and four and a half times greater during NREM sleep compared with the C57BL/6J-Lep^ob mice. The differences in response to CO₂ challenge were also reflected in greater increases (p < 0.001) in the frequency responses (Table 2) in the wild-type mice compared with the C57BL/6J-Lep^ob mice.

View larger version (22K): [in this window] [in a new window]   Figure 3.   Minute ventilation ( E, left panel) at increasing levels of inspired CO2 and the hypercapnic ventilatory response (HCVR, right panel) across sleep/wake states are compared between female wild-type C57BL/6J mice fed a regular diet, wild-type C57BL/6J mice fed a high fat diet, and C57BL/6J-Lepob mice. Results presented show the mean ± SEM. Significance between groups was derived within each sleep/wake state using one-way, within-subject ANOVA.

High fat diet C57BL/6J. We examined the effect of obesity on respiratory control in wild-type mice fed a high fat diet. Although the female wild-type mice fed the high fat diet could not attain the weights of the C57BL/6J-Lep^ob mice, they did increase their body weight by 66% compared with the female wild-type mice on the regular diet (Table 1). Obesity in the female wild-type mice did not result in decrements in ventilatory responses to CO₂ challenge compared with the female mice on the regular diet (Figure 3). Indeed, during NREM sleep the HCVR of the high fat diet female wild-type mice (9.4 ± 0.4 ml/min/% CO₂) was even higher (p < 0.03) than the female wild-type mice on the regular diet.

Wild-type sex differences in E and the HCVR. There was no significant difference in baseline E between male and female wild-type mice across all sleep/wake states, both for the regular diet and high fat diet groups. For female mice on a high fat diet, baseline E was 61.3 ± 2.7 ml/min during wakefulness, 53.5 ± 2.6 ml/min during NREM sleep, and 62.6 ± 3.8 ml/min during REM sleep in comparison with 59.6 ± 3.9 ml/min, 56.6 ± 3.2 ml/min, and 59.2 ± 4.8 ml/min, respectively, in male mice. The respiratory drive (VT/TI) was also identical between the groups of male and female wild-type mice during wakefulness and NREM sleep (Table 3).

We also compared the HCVR between male and female wild-type mice. When the regular diet and the high fat diet groups were analyzed separately, there was no statistical effect of sex between the HCVR during wakefulness and NREM sleep, although a trend existed for the HCVR during REM sleep to be higher in males compared with females. When data from the regular diet and the high fat diet groups were pooled, the HCVR during REM sleep was double (p < 0.05) in male compared with female wild-type mice (Figure 4).

View larger version (14K): [in this window] [in a new window]   Figure 4.   The hypercapnic ventilatory response (HCVR) in male and female wild-type C57BL/6J mice on both regular and high fat diets across sleep/wake states. Results presented show the mean ± SEM. Significance between groups was derived within each sleep/wake state using one-way, within-subject ANOVA.

	DISCUSSION

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Past studies have examined the effect of either obesity or sex on respiration (2, 3, 8, 9, 11, 23), but few data are available on how sex and obesity may interact to affect respiratory control. In general, females are considered less likely than males to exhibit respiratory depression because of the presence of higher levels of the respiratory stimulant progesterone (7, 11). In the present study, we confirm our previous work showing that the absence of leptin results in severe obesity and respiratory depression. We now extend this work to examine the interaction of sex and obesity and demonstrate that respiratory depression in female C57BL/6J-Lep^ob mice is even more severe than in weight-matched male C57BL/6J-Lep^ob mice. These changes in respiratory control could not be attributed to sex steroids because the levels of progesterone and estradiol were comparable between obese female and male C57BL/6J-Lep^ob mice. Sex per se could also not account for the severe respiratory depression in female C57BL/6J-Lep^ob mice, as both normal weight and obese female wild-type mice exhibited ventilatory responses to CO₂ that were comparable to weight-matched male wild-type mice. Finally during both NREM and REM sleep, ventilatory responses to CO₂ were barely detectable in female C57BL/6J-Lep^ob mice, indicating blunted ventilatory responses across all stages of sleep. Thus, the unique interaction of female sex, obesity, and leptin deficiency results in a profound degree of respiratory depression that is further exacerbated during sleep.

Our mouse model has several advantages for exploring the relationship between sex, obesity, and respiratory control. First, the animals are highly inbred making the HCVR extremely reproducible between animals. In contrast, considerable individual variability exists in measurements of ventilatory responses to CO₂ challenge in humans (24). Consequently, no clear consensus exists on whether sex affects the HCVR, with responses being reported as either lower (26) or higher (27) in females compared with males. Second, mice do not exhibit obstructive sleep apnea, even in the presence of severe obesity, allowing us to examine effects of obesity on respiratory control independent of any confounding influences of upper airway occlusion. Third, considerable changes in the HCVR are known to occur across the sleep/wake state (16). We have, therefore, developed techniques to simultaneously record polysomnography and barometric plethysmography that have allowed us to compare the HCVR in specific sleep/ wake states. Thus, our observations regarding the impact of leptin deficiency on respiratory control in inbred female mice may provide insights that are not possible even in carefully controlled human studies.

Previous investigators who have examined effects of sex on respiratory control have focused primarily on the role of female sex steroids (11). For example, progesterone can influence respiratory control in a number of ways. First, progesterone can increase chemosensitivity peripherally, increasing carotid body neural output in response to hypoxia, as well as within the central nervous system (CNS) (11). Second, elevated progesterone levels associated with pregnancy can increase resting minute ventilation and ventilatory responses to CO₂ (12). Third, progesterone may protect the upper airway during sleep leading to a lower prevalence of obstructive sleep apnea among women (9). In our study, however, the effects of leptin deficiency in relationship to sex were independent of differences in female sex steroids. We cannot, therefore, attribute our observed differences in respiratory control to sex steroids, indicating that some other sex-related factor(s) must be involved in the interaction of female sex, obesity, and leptin deficiency on respiratory depression.

Our finding that respiratory control was markedly impaired in female C57BL/6J-Lep^ob mice could be potentially related to sex per se rather than leptin deficiency. To test this possibility, we examined ventilatory responses in weight-matched female and male wild-type mice on either a regular diet or a high fat diet. In contrast to female C57BL/6J-Lep^ob mice, ventilatory responses in female wild-type mice during wakefulness and NREM sleep were identical to male wild-type mice. Furthermore, diet-induced obesity in female wild-type mice did not appear to negatively impact ventilatory responses. In fact, during NREM sleep the obese female wild-type mice demonstrated a significantly elevated HCVR compared with regular weight female wildtype mice. Thus, we have no evidence that sex alone accounts for the severe respiratory depression in female C57BL/6J-Lep^ob mice.

Although sex per se does not influence respiratory control, we confirmed that leptin levels were higher in female wild-type mice on both a regular diet and a high fat diet than weight-matched male wild-type mice on comparable diets (Table 1). We speculate that the elevated circulating leptin levels in the female wild-type mice were necessary to preserve respiratory control at levels equivalent to weight-matched wild-type male mice with lower leptin levels. Consequently, if female and male wild-type mice of comparable weight were to have comparable leptin levels we would predict a greater degree of respiratory depression in the female mice. In the extreme, complete leptin deficiency and severe obesity would represent the greatest risk for female mice, as demonstrated in female C57BL/6J-Lep^ob mice in the present study.

As can be seen (Table 3), the severe respiratory depression in female C57BL/6J-Lep^ob mice was the result of an impaired ventilatory drive during hypercapnic challenge. For example, the increase in VT/TI was 2- to 3-fold less in female versus male C57BL/6J-Lep^ob mice during 8% CO₂ challenge. In contrast, respiratory frequency responses to CO₂ challenge were comparable between female and male C57BL/6J-Lep^ob mice, despite overall lower frequency in the females. These data suggest that ventilatory drive is particularly sensitive to the absence of leptin in female C57BL/6J-Lep^ob mice. Interestingly, we reported in a previous study that leptin replacement in male C57BL/6J-Lep^ob mice increased ventilatory responses to CO₂ challenge by augmenting tidal volume rather than frequency responses during both wakefulness and NREM sleep (16). It is consistent, therefore, that impairment of ventilatory drive is the predominant factor inducing respiratory depression in obese female mice that lack leptin.

It should be noted that the most significant impact of leptin deficiency on ventilatory control occurs in REM sleep. We have previously shown that male C57BL/6J-Lep^ob mice are incapable of increasing their ventilation in response to hypercapnic challenge during REM sleep. Furthermore, leptin replacement in male C57BL/6J-Lep^ob mice caused substantial increases in baseline minute ventilation and restored a significant HCVR during REM sleep (16). In the current study, we have shown leptin-deficient female C57BL/6J-Lep^ob mice also exhibit a flat HCVR during REM sleep. Surprisingly, female wild-type mice exhibited an HCVR during REM sleep that was significantly depressed compared with male wild-type mice. Thus, REM sleep may unmask a susceptibility for respiratory depression in females even in the presence of normal or elevated leptin levels.

There are several potential clinical manifestations that can be drawn from our work. Clearly, if the ob/ob phenotype was not rare in humans, such leptin deficiency in the presence of obesity would lead to profound clinical problems. However, human obesity is normally associated with elevated leptin levels (18) and this has led us to hypothesize that a relative leptin deficiency in the CNS or a leptin "resistance" may predispose to obesity hypoventilation syndrome (OHS). Accordingly, the much higher endogenous levels of leptin in females would appear to play a protective role against the development of OHS. In humans (17, 18) and in wild-type mice (Table 1) the leptin level increases in proportion to the degree of adiposity, but leptin levels are significantly elevated in females compared with males (17, 19, 20, 29). Differences in fat distribution between females and males may account for higher leptin levels in females (17, 19, 29). Subcutaneous fat, which is more prevalent in females, produces more leptin than abdominal fat, which is more prevalent in males (19). It is unclear whether the sex differences in leptin levels in mice are also due to differences in fat distribution or to some other factor(s) related to sex. Whatever the mechanism, it is clear that C57BL/6J mice can duplicate the sex and obesity effects on leptin levels reported for humans. Clinical studies have shown that cerebro spinal fluid (CSF), as well as serum, leptin levels (30) are higher in females compared with males. These human data, taken together with our current results, indicate that elevated CNS leptin levels in females may be necessary just to maintain respiratory control characteristics comparable to males, and even this may not be sufficient during susceptible periods of REM sleep. Thus, low CNS leptin levels in the presence of obesity may represent a high risk for the development of OHS in females.