Respiratory functions affected by Leptins
Leptin is a protein produced by adipose tissues that circulate in the brain and interact with the receptors in the hypothalamus to inhibit eating. Importance of this peptide is demonstrated by profound obesity exhibited by the ob/ob mouse (C57BL/6J-Lep(ob)) which is unable to produce functional leptin. The measurement of respiratory functions in this mouse shows that profound obesity is associated with impaired respiratory mechanics and depressed respiratory control, particularly during sleep. Leptin may act as growth factor in the lung as well as neuro-hormonal modulator of central respiratory control mechanisms. Human obesity is associated with more variable leptin levels for a given degree of adiposity. Thus, relative deficiency in leptin, or a leptin resistance, may play a role in obesity-related breathing disorders such as obesity hypoventilation syndrome (OHS) or obstructive sleep apnea (OSA).
Respiratory functions affected by leptins
Obesity has taken a form of epidemic in several developed countries and it is continuously rising (Mokdad AH et al. 2001). Several diseases are associated with the obesity, one of most important of which is respiratory function impairment. It can cause airway depression and impaired activity of upper airway tract muscles especially during sleep can cause obstructive sleep apnoea in >50% of obese patients (body mass index >30 kg/m2) (Punjabi NM et al. 2002) whereas severe obesity (body mass index >40 kg/m2) can cause depression of the respiratory pump muscles during wakefulness, resulting in CO2 retention and the syndrome of obesity hypoventilation (OHS) (Berger KI et al. 2001). Similar phenomenon has been observed in animal models of obesity such as the ob mouse (C57BL/6J-Lepob). It is proposed that a constellation of respiratory complications are attenuated with leptin (i.e. protein product of the ob gene) replacement.
Leptin is a satiety producing hormone secreted by adipocytes (Friedman JM and Halaas JL 1998) that provides a common link between obesity and respiratory depression. Initial study demonstrated that complete impairment of leptin signaling in genetically obese ob/ob mice significantly reduced the ventilatory response to hypercapnia during wakefulness (Tankersley C, Kleeberger S, Russ B, Schwartz A, and Smith P 1996). Subsequent clinical studies have shown that both OSA and OHS are associated with the most common form of impaired leptin signaling in humans, namely leptin resistance (Ip MS, Lam KS, Ho C, Tsang KW, and Lam W. et al. 2000).
Thus animal and human studies both shows that leptin resistance or deficiency may cause depressed hypercapnic sensitivity especially during sleep. Central effect of leptin on satiety and metabolism is predominantly through arcuate nucleus in hypothalamus. The anorexigenic effects of leptin occur primarily through activation of melanocortin 4 (MC4) receptors and inhibition of neuropeptide Y (NPY) in the hypothalamus. Putatively, MC4 and NPY pathways could impact on respiratory control via neural connections between the hypothalamus and respiratory control centers in the medulla (Glass MJ, Chan J, and Pickel VM 2002).Thus impairment of MC4 pathways or activation of NPY pathways in the hypothalamus could contribute to the respiratory depression associated with impaired leptin signaling.
The combination of leptin deficiency and profound weight gain in adult ob/ob mice can produce marked changes in respiratory mechanics (Tankersley, C.G., O'Donnell, C. and Daood, M.J., 1998. ). Ob/ob mouse shows that the profound obesity is associated with impaired respiratory mechanics and depressed respiratory control, particularly during sleep. Moreover, wild type mice with diet-induced obesity have normal respiratory function associated with markedly elevated leptin levels. Human obesity, similar to obesity in wild type mice, also causes an elevation in circulating leptin. But it does not show any tight relations, it is moreover variable in nature.
Methodology
Animals- Twelve mutant obese C57BL/6J-Lepob male mice, 22 C57BL/6J male mice, 7 mutant Ay male mice from Jackson Laboratory (Bar Harbor, ME), 9 NPY-deficient transgenic male mice (NPY-/-), and 9 littermates (NPY+/+),were used in this study.
Sleep/wake state was assessed from EEG and EMG recordings. Wakefulness is characterized by low-amplitude high-frequency (10-20 Hz) EEG waves and high levels of EMG activity as compare to sleep states. Non-rapid eye movement (NREM) sleep is characterized by high-amplitude, low-frequency (2-5 Hz) EEG waves and an EMG activity less than during wakefulness. Rapid eye movement (REM) sleep was characterized by low-amplitude; mixed-frequency (5-10 Hz) EEG waves, although the predominant pattern was a fixed amplitude theta frequency consistent with hippocampal theta rhythm. During REM sleep, the EMG activity was either equal to or less than that seen during NREM sleep but was always less than that seen during wakefulness.
Ventilation was measured during wakefulness and 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) and hypoxic gases (15% O2 and 10% O2 in 3% CO2 to eliminate hypoxia-induced hypocapnia). Least squares linear regression analysis was used to calculate HCVR (slope of the relationship between E and inspired CO2) and HVR (slope of the relationship between E and inspired O2) during wakefulness and NREM sleep. Data for the HVR are not presented during REM sleep due to the absence of sustained periods of REM sleep during hypoxic exposure.
Experimental groups-Comparisons of ventilatory control parameters were made between mice in three separate series of experiments: 1) wild-type lean C57BL/6J mice of 30 g of weight maintained on a regular ad libitum chow diet (WT30), wild-type C57BL/6J mice maintained on a high-fat diet (49% fat; 5.8 kcal/g) for 16 wk to develop diet-induced obesity with 40 g of weight (WT40), C57BL/6J-Lepob mice of 40 g of weight maintained on a regular ad libitum chow diet (OB40), and C57BL/6J-Lepob mice of 60 g of weight maintained on a regular ad libitum chow diet (OB60); 2) NPY-/- and NPY+/+ mice maintained on a regular ad libitum chow diet; and 3) Ay and weight-matched wild-type C57BL/6J mice maintained on a high-fat diet (Ay control).
Plasma leptin determination-Arterial blood (1-1.2 ml) was obtained from direct cardiac puncture under iso-flurane anesthesia. Serum leptin levels were measured with a mouse leptin radioimmunoassay kit from Linco Research (St. Charles, MO).
Statistical analyses- Data were analyzed by using Crunch 4 (Crunch Software; Oakland, CA), and results for E, HCVR, and HVR are shown as means ± SE. Statistical significance between groups was derived within each sleep/wake state by using ANOVA with Newman-Keuls post hoc analyses where appropriate.
Results
VE and HVR in leptin-deficient mice- In the OB40 group, baseline VE had a trend to be lower than in weight-matched obese wild-type mice (WT40 group) during wakefulness (P = 0.06) and was significantly lower during NREM sleep (37.5 ± 2.7 vs. 55.8 ± 3.1 ml/min, P < 0.05; and REM sleep (36.4 ± 3.6 vs. 61.2 ± 3.8 ml/min, P < 0.05). This difference in baseline VE between the OB40 and WT40 strain was entirely due to a significantly lower VT in the leptin deficient mice. The OB60 mice had significantly higher levels of VE at baseline than OB40 mice due to larger VT across all sleep/wake stages . However, when VE was corrected per body weight, there was no difference between the two groups. Similarly, in C57BL/6J mice, VE and VT were higher in the WT40 mice than in the WT30 mice, and the difference was no longer present when VE was corrected per body weight. There was no statistically significant difference in the HVR or hypoxia-induced changes in VT and f during either wakefulness or NREM sleep between all four groups of mice, regardless of the presence or absence of leptin deficiency and obesity.
HVR and HCVR in NPY-deficient mice - Body weight and serum leptin levels (2.1 ± 0.1 vs. 2.6 ± 0.2 ng/ml) were similar between NPY-/- and NPY+/+ mice. There was no significant difference in either baseline respiratory frequency, VT, E, or the HVR between the two strains across all sleep/wake states. During hypercapnic challenge in wakefulness, the NPY-/- animals exhibited an elevated HCVR (P < 0.05) compared with wild-type littermates due to a trend to a larger increase in VT. There was no difference in the HCVR between the two groups of mice in NREM and REM sleep.
HVR and HCVR in Ay mice - The elevated body weight in both Ay and control mice was associated with high leptin levels that tended to be greater in Ay (29.0 ± 4.9 ng/ml) than in control mice (21.2 ± 1.3 ng/ml) but did not reach statistical significance (P = 0.13). Baseline VE was significantly lower in Ay mice compared with control mice across all sleep/wake stages . Low levels of E in Ay mice could be attributed to both smaller VT and f than in control animals. The HVR was identical in both strains. During NREM sleep, Ay mice exhibited a significant depression of the HCVR compared with control mice, which was entirely due to smaller increases in VT. During wakefulness and REM sleep, the HCVR was similar between strains ( Vsevolod Y. Polotsky et al.2004).
There are several outcomes of this study. This study shows relationship between levels of leptin and interruption of leptin signaling pathways (MC4 and NPY) on respiratory control. Some new findings which are concluded from this study are baseline VE is decreased in both leptin-deficient mice and Ay mice with disrupted MC4 pathways but not in NPY-/- mice. This reduction in baseline VE in leptin-deficient and Ay mice is likely due to the reduced basal metabolic rate reported in these strains (Breslow MJ et al. 1999).
Second-the HCVR (hypercapnic ventilatory responsiveness), which is depressed by leptin deficiency (O'Donnell CP et al.1998) and restored by leptin replacement in ob/ob mice during sleep, is also depressed by the MC4 blockade in Ay mice during NREM sleep.
Third, the HVR (hypoxic ventilatory response) is not affected by leptin deficiency, MC4 blockade, or NPY deficiency, suggesting that 1) the effect of leptin on ventilatory control is specific for CO2-sensing neurons located in medulla and 2) leptin deficiency does not impact on the sensing of hypoxia either centrally by the medullary receptors or peripherally by the carotid bodies.
Baseline VE-It is confirmed from this study that leptin deficiency is associated with decrease in baseline VE more during sleep as compare to wakefulness. It is due to centrally mediated reduction in metabolic rate present in the leptin-deficient mice. It also proves that action of leptin is mediated via both MC4 and NPY pathways (Erickson JC, Hollopeter G, and Palmiter RD 1996).
Central leptin signaling pathways and respiratory control-leptin deficiency leads to marked suppression of the HCVR. Leptin replacement caused large increases in baseline VE and significantly increased the HCVR during NREM and REM sleep, independent of weight, metabolism, and food intake.
Leptin and human respiratory control
Human respiratory control and leptin relationship is different to that found in ob/ob mice. Based on the findings in mice obese individuals can be on risk by two mechanisms-
when plasma and CSF leptin levels are low (Caro and Schwartz 1996);
when plasma leptin levels are high, but CSF leptin levels are proportionately low
The ob/ob mouse reproduces the primary clinical features of OHS. A clear rationale exists for investigating the relationship between leptin and the respiratory pump muscles in obese humans. In OSA, sleep is the factor that predisposes the upper airway to collapse (Gastaut et al., 1965). As noted above, the effects of leptin on ventilatory control in mice were more pronounced during sleep than wakefulness ( O’Donnell et al., 1999). Assuming that leptin can influence the upper airway in sleeping humans, this would suggest that the presence of elevated leptin levels in the CNS might protect against OSA.
It is noted that absence of leptin in ob/ob mice does not cause observable collapse of upper airway but it does not exclude the possibility of leptin relation with airway in humans because of three reasons. First, the minute cross-sectional area of the mouse upper airway may produce mechanical stability, since, according to the law of Laplace, the wall tension required to maintain a given trans-mural pressure decreases in proportion to the radius.
Second, the neuronal circuitry controlling upper airway collapsibility (upper airway muscles) or CO2 retention (diaphragmatic pump muscles) may differ between mice and humans. Third, the distribution of leptin receptors in humans in areas of the CNS controlling upper airway and pump muscles may not be comparable to that of the mouse. Thus relationship between leptin levels and respiration in humans is a matter of further research.
It is also noted that gender plays an important role in development of sleep apnoea disorders. In women, for the same BMI incidence of sleep apnoea is lower than that of males with same leptin levels. This may be explained by presence of different sex hormones that might play a protective role in development of sleep disorders. (Camargo et al. 1998).
In summary above study shows a relationship between leptin levels and respiratory control. Insufficient leptin levels in obese individuals may cause Obesity hypo-ventilation syndrome. This effect of leptin is more profound during sleep, which shows that it plays a role in development of sleep apnea disorders. Leptin deficiency control on respiration is specific to hypercapnia only and is not related to hypoxia. Thus leptin deficiency does not impact on central or peripheral chemoceptors that senses hypoxia. It is also concluded that effect of leptin on respiration is dependent on hypothalamic pathway. Leptin receptors are also abundant in the nucleus of the solitary tract and other centres in the medulla involved in respiratory responses to CO2 and pH. Human obesity is most commonly related with elevated leptin levels called as leptin resistance. Thus leptin resistance in obese individuals can cause sleep apnoea.
Respiratory functions affected by leptins
Obesity has taken a form of epidemic in several developed countries and it is continuously rising (Mokdad AH et al. 2001). Several diseases are associated with the obesity, one of most important of which is respiratory function impairment. It can cause airway depression and impaired activity of upper airway tract muscles especially during sleep can cause obstructive sleep apnoea in >50% of obese patients (body mass index >30 kg/m2) (Punjabi NM et al. 2002) whereas severe obesity (body mass index >40 kg/m2) can cause depression of the respiratory pump muscles during wakefulness, resulting in CO2 retention and the syndrome of obesity hypoventilation (OHS) (Berger KI et al. 2001). Similar phenomenon has been observed in animal models of obesity such as the ob mouse (C57BL/6J-Lepob). It is proposed that a constellation of respiratory complications are attenuated with leptin (i.e. protein product of the ob gene) replacement.
Leptin is a satiety producing hormone secreted by adipocytes (Friedman JM and Halaas JL 1998) that provides a common link between obesity and respiratory depression. Initial study demonstrated that complete impairment of leptin signaling in genetically obese ob/ob mice significantly reduced the ventilatory response to hypercapnia during wakefulness (Tankersley C, Kleeberger S, Russ B, Schwartz A, and Smith P 1996). Subsequent clinical studies have shown that both OSA and OHS are associated with the most common form of impaired leptin signaling in humans, namely leptin resistance (Ip MS, Lam KS, Ho C, Tsang KW, and Lam W. et al. 2000).
Thus animal and human studies both shows that leptin resistance or deficiency may cause depressed hypercapnic sensitivity especially during sleep. Central effect of leptin on satiety and metabolism is predominantly through arcuate nucleus in hypothalamus. The anorexigenic effects of leptin occur primarily through activation of melanocortin 4 (MC4) receptors and inhibition of neuropeptide Y (NPY) in the hypothalamus. Putatively, MC4 and NPY pathways could impact on respiratory control via neural connections between the hypothalamus and respiratory control centers in the medulla (Glass MJ, Chan J, and Pickel VM 2002).Thus impairment of MC4 pathways or activation of NPY pathways in the hypothalamus could contribute to the respiratory depression associated with impaired leptin signaling.
The combination of leptin deficiency and profound weight gain in adult ob/ob mice can produce marked changes in respiratory mechanics (Tankersley, C.G., O'Donnell, C. and Daood, M.J., 1998. ). Ob/ob mouse shows that the profound obesity is associated with impaired respiratory mechanics and depressed respiratory control, particularly during sleep. Moreover, wild type mice with diet-induced obesity have normal respiratory function associated with markedly elevated leptin levels. Human obesity, similar to obesity in wild type mice, also causes an elevation in circulating leptin. But it does not show any tight relations, it is moreover variable in nature.
Methodology
Animals- Twelve mutant obese C57BL/6J-Lepob male mice, 22 C57BL/6J male mice, 7 mutant Ay male mice from Jackson Laboratory (Bar Harbor, ME), 9 NPY-deficient transgenic male mice (NPY-/-), and 9 littermates (NPY+/+),were used in this study.
Sleep/wake state was assessed from EEG and EMG recordings. Wakefulness is characterized by low-amplitude high-frequency (10-20 Hz) EEG waves and high levels of EMG activity as compare to sleep states. Non-rapid eye movement (NREM) sleep is characterized by high-amplitude, low-frequency (2-5 Hz) EEG waves and an EMG activity less than during wakefulness. Rapid eye movement (REM) sleep was characterized by low-amplitude; mixed-frequency (5-10 Hz) EEG waves, although the predominant pattern was a fixed amplitude theta frequency consistent with hippocampal theta rhythm. During REM sleep, the EMG activity was either equal to or less than that seen during NREM sleep but was always less than that seen during wakefulness.
Ventilation was measured during wakefulness and 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) and hypoxic gases (15% O2 and 10% O2 in 3% CO2 to eliminate hypoxia-induced hypocapnia). Least squares linear regression analysis was used to calculate HCVR (slope of the relationship between E and inspired CO2) and HVR (slope of the relationship between E and inspired O2) during wakefulness and NREM sleep. Data for the HVR are not presented during REM sleep due to the absence of sustained periods of REM sleep during hypoxic exposure.
Experimental groups-Comparisons of ventilatory control parameters were made between mice in three separate series of experiments: 1) wild-type lean C57BL/6J mice of 30 g of weight maintained on a regular ad libitum chow diet (WT30), wild-type C57BL/6J mice maintained on a high-fat diet (49% fat; 5.8 kcal/g) for 16 wk to develop diet-induced obesity with 40 g of weight (WT40), C57BL/6J-Lepob mice of 40 g of weight maintained on a regular ad libitum chow diet (OB40), and C57BL/6J-Lepob mice of 60 g of weight maintained on a regular ad libitum chow diet (OB60); 2) NPY-/- and NPY+/+ mice maintained on a regular ad libitum chow diet; and 3) Ay and weight-matched wild-type C57BL/6J mice maintained on a high-fat diet (Ay control).
Plasma leptin determination-Arterial blood (1-1.2 ml) was obtained from direct cardiac puncture under iso-flurane anesthesia. Serum leptin levels were measured with a mouse leptin radioimmunoassay kit from Linco Research (St. Charles, MO).
Statistical analyses- Data were analyzed by using Crunch 4 (Crunch Software; Oakland, CA), and results for E, HCVR, and HVR are shown as means ± SE. Statistical significance between groups was derived within each sleep/wake state by using ANOVA with Newman-Keuls post hoc analyses where appropriate.
Results
VE and HVR in leptin-deficient mice- In the OB40 group, baseline VE had a trend to be lower than in weight-matched obese wild-type mice (WT40 group) during wakefulness (P = 0.06) and was significantly lower during NREM sleep (37.5 ± 2.7 vs. 55.8 ± 3.1 ml/min, P < 0.05; and REM sleep (36.4 ± 3.6 vs. 61.2 ± 3.8 ml/min, P < 0.05). This difference in baseline VE between the OB40 and WT40 strain was entirely due to a significantly lower VT in the leptin deficient mice. The OB60 mice had significantly higher levels of VE at baseline than OB40 mice due to larger VT across all sleep/wake stages . However, when VE was corrected per body weight, there was no difference between the two groups. Similarly, in C57BL/6J mice, VE and VT were higher in the WT40 mice than in the WT30 mice, and the difference was no longer present when VE was corrected per body weight. There was no statistically significant difference in the HVR or hypoxia-induced changes in VT and f during either wakefulness or NREM sleep between all four groups of mice, regardless of the presence or absence of leptin deficiency and obesity.
HVR and HCVR in NPY-deficient mice - Body weight and serum leptin levels (2.1 ± 0.1 vs. 2.6 ± 0.2 ng/ml) were similar between NPY-/- and NPY+/+ mice. There was no significant difference in either baseline respiratory frequency, VT, E, or the HVR between the two strains across all sleep/wake states. During hypercapnic challenge in wakefulness, the NPY-/- animals exhibited an elevated HCVR (P < 0.05) compared with wild-type littermates due to a trend to a larger increase in VT. There was no difference in the HCVR between the two groups of mice in NREM and REM sleep.
HVR and HCVR in Ay mice - The elevated body weight in both Ay and control mice was associated with high leptin levels that tended to be greater in Ay (29.0 ± 4.9 ng/ml) than in control mice (21.2 ± 1.3 ng/ml) but did not reach statistical significance (P = 0.13). Baseline VE was significantly lower in Ay mice compared with control mice across all sleep/wake stages . Low levels of E in Ay mice could be attributed to both smaller VT and f than in control animals. The HVR was identical in both strains. During NREM sleep, Ay mice exhibited a significant depression of the HCVR compared with control mice, which was entirely due to smaller increases in VT. During wakefulness and REM sleep, the HCVR was similar between strains ( Vsevolod Y. Polotsky et al.2004).
There are several outcomes of this study. This study shows relationship between levels of leptin and interruption of leptin signaling pathways (MC4 and NPY) on respiratory control. Some new findings which are concluded from this study are baseline VE is decreased in both leptin-deficient mice and Ay mice with disrupted MC4 pathways but not in NPY-/- mice. This reduction in baseline VE in leptin-deficient and Ay mice is likely due to the reduced basal metabolic rate reported in these strains (Breslow MJ et al. 1999).
Second-the HCVR (hypercapnic ventilatory responsiveness), which is depressed by leptin deficiency (O'Donnell CP et al.1998) and restored by leptin replacement in ob/ob mice during sleep, is also depressed by the MC4 blockade in Ay mice during NREM sleep.
Third, the HVR (hypoxic ventilatory response) is not affected by leptin deficiency, MC4 blockade, or NPY deficiency, suggesting that 1) the effect of leptin on ventilatory control is specific for CO2-sensing neurons located in medulla and 2) leptin deficiency does not impact on the sensing of hypoxia either centrally by the medullary receptors or peripherally by the carotid bodies.
Baseline VE-It is confirmed from this study that leptin deficiency is associated with decrease in baseline VE more during sleep as compare to wakefulness. It is due to centrally mediated reduction in metabolic rate present in the leptin-deficient mice. It also proves that action of leptin is mediated via both MC4 and NPY pathways (Erickson JC, Hollopeter G, and Palmiter RD 1996).
Central leptin signaling pathways and respiratory control-leptin deficiency leads to marked suppression of the HCVR. Leptin replacement caused large increases in baseline VE and significantly increased the HCVR during NREM and REM sleep, independent of weight, metabolism, and food intake.
Leptin and human respiratory control
Human respiratory control and leptin relationship is different to that found in ob/ob mice. Based on the findings in mice obese individuals can be on risk by two mechanisms-
when plasma and CSF leptin levels are low (Caro and Schwartz 1996);
when plasma leptin levels are high, but CSF leptin levels are proportionately low
The ob/ob mouse reproduces the primary clinical features of OHS. A clear rationale exists for investigating the relationship between leptin and the respiratory pump muscles in obese humans. In OSA, sleep is the factor that predisposes the upper airway to collapse (Gastaut et al., 1965). As noted above, the effects of leptin on ventilatory control in mice were more pronounced during sleep than wakefulness ( O’Donnell et al., 1999). Assuming that leptin can influence the upper airway in sleeping humans, this would suggest that the presence of elevated leptin levels in the CNS might protect against OSA.
It is noted that absence of leptin in ob/ob mice does not cause observable collapse of upper airway but it does not exclude the possibility of leptin relation with airway in humans because of three reasons. First, the minute cross-sectional area of the mouse upper airway may produce mechanical stability, since, according to the law of Laplace, the wall tension required to maintain a given trans-mural pressure decreases in proportion to the radius.
Second, the neuronal circuitry controlling upper airway collapsibility (upper airway muscles) or CO2 retention (diaphragmatic pump muscles) may differ between mice and humans. Third, the distribution of leptin receptors in humans in areas of the CNS controlling upper airway and pump muscles may not be comparable to that of the mouse. Thus relationship between leptin levels and respiration in humans is a matter of further research.
It is also noted that gender plays an important role in development of sleep apnoea disorders. In women, for the same BMI incidence of sleep apnoea is lower than that of males with same leptin levels. This may be explained by presence of different sex hormones that might play a protective role in development of sleep disorders. (Camargo et al. 1998).
In summary above study shows a relationship between leptin levels and respiratory control. Insufficient leptin levels in obese individuals may cause Obesity hypo-ventilation syndrome. This effect of leptin is more profound during sleep, which shows that it plays a role in development of sleep apnea disorders. Leptin deficiency control on respiration is specific to hypercapnia only and is not related to hypoxia. Thus leptin deficiency does not impact on central or peripheral chemoceptors that senses hypoxia. It is also concluded that effect of leptin on respiration is dependent on hypothalamic pathway. Leptin receptors are also abundant in the nucleus of the solitary tract and other centres in the medulla involved in respiratory responses to CO2 and pH. Human obesity is most commonly related with elevated leptin levels called as leptin resistance. Thus leptin resistance in obese individuals can cause sleep apnoea.