Effects of Insufficient Sleep on Pituitary–Adrenocortical Response to CRH Stimulation in Healthy Men

Abstract

Study Objectives:

Severe sleep restriction results in elevated evening cortisol levels. We examined whether this relative hypercortisolism is associated with alterations in the pituitary–adrenocortical response to evening corticotropin-releasing hormone (CRH) stimulation.

Methods:

Eleven subjects participated in 2 sessions (2 nights of 10 hours vs. 4 hours in bed) in randomized order. Sleep was polygraphically recorded. After the second night of each session, blood was sampled at 20-minute intervals from 09:00 to 24:00 for adrenocorticotropic hormone (ACTH) and cortisol measurements, and perceived stress was assessed hourly. Ovine CRH was injected at 18:00 (1 µg/kg body weight).

Results:

Prior to CRH injection, baseline ACTH, but not cortisol, levels were elevated after sleep restriction. Relative to the well-rested condition, sleep restriction resulted in a 27% decrease in overall ACTH response to CRH (estimated by the incremental area under the curve from 18:00 to 24:00; p = .002) while the cortisol response was decreased by 21% (p = .083). Further, the magnitude of these decreases was correlated with the individual amount of sleep loss (ACTH: r_Sp = −0.65, p = .032; cortisol: r_Sp = −0.71, p = .015). The acute post-CRH increment of cortisol was reduced (p = .002) without changes in ACTH reactivity, suggesting decreased adrenal sensitivity. The rate of decline from peak post-injection levels was reduced for cortisol (p = .032), but not for ACTH. Scores of perceived stress were unaffected by CRH injection and were low and similar under both sleep conditions.

Conclusions:

Sleep restriction is associated with a reduction of the overall ACTH and cortisol responses to evening CRH stimulation, and a reduced reactivity and slower recovery of the cortisol response.

Statement of Significance

The findings of this study indicate that the impact of short-term severe sleep restriction on hypothalamo–pituitary–adrenal (HPA) axis activity includes an increase in basal ACTH levels, blunted overall ACTH and cortisol responses to CRH stimulation as well as a reduced reactivity and recovery of the acute cortisol response to CRH, a pivotal neuroendocrine transducer of the physiologic response to stress. These abnormalities in HPA function may mediate the association between insufficient sleep and increased risk for various conditions, including obesity, diabetes, cardiovascular disease, and vulnerability to infection.

INTRODUCTION

Behavioral sleep curtailment is common in modern societies. While the American National Sleep Foundation recommends 7–9 hours of sleep for adults,¹ roughly 1/3 of American,² French,³ and British⁴ adults report sleeping ≤ 6 hours on a regular basis, and 7% of British adults report sleep times of less than 5 hours.⁴ Although, self-reported sleep time do not agree with actual sleep,⁵ objectively recorded sleep of less than 6 hours per night has been reported for 43% of middle-aged adults in the CARDIA cohort Sleep Study⁶ and average sleep times of 3.8 h/day have been recorded in truck drivers.⁷

In the past two decades, evidence has accumulated to indicate that one of the hormonal consequences of insufficient sleep loss is a relative state of hypercortisolism in the later part of the day. Indeed, in a number of well-controlled laboratory studies of healthy participants, sleep loss, resulting from either acute total sleep deprivation or severe recurrent sleep restriction (≤5, 5 h/night) resulted in elevated evening cortisol levels, often associated with a slower decline of cortisol concentrations across the day.^8–17 However, some studies,^18–20 and especially those using less severe sleep deprivation paradigms (time in bed ≥ 6 hours^{19, 20}), found no evidence of increased evening cortisol levels following sleep deprivation. Interestingly, in well-documented cross-sectional²¹ and longitudinal²² epidemiologic studies, chronic short sleepers were found to be at higher risk of elevated evening cortisol levels.

It was initially hypothesized that this elevation of evening cortisol concentration in a state of sleep debt reflected a slower rate of recovery of the hypothalamo–pituitary–adrenal (HPA) axis from the circadian-driven morning stimulation, that is, impaired glucocorticoid negative feedback,^8,9 rather than a direct stimulatory effect of sleep deprivation on HPA activity. Animal studies, however, have pointed towards a direct stimulatory effect of sleep loss on the HPA axis.^23–25 Similarly, a recent study in healthy humans reported elevated daytime ACTH levels following 2 nights of 4 hours in bed, therefore providing evidence for an up-regulation of the HPA axis during severe sleep loss in humans.¹⁶ Under chronic conditions, the relationship between HPA axis over-activity and disturbed sleep may be bidirectional as elevated evening cortisol levels are likely to disturb sleep.^26,27 As such, insufficient sleep and elevated cortisol may be involved in a feed forward cascade of negative events with long-term adverse health consequences.²⁷ Additionally, a direct consequence of an evening elevation of cortisol levels is a dampening of the 24-hour rhythm of glucocorticoid levels, a powerful internal synchronizer of central and peripheral circadian clocks.^28,29 Circadian misalignment has recently been associated with a host of adverse effects.^28,29

Studies in rodents have demonstrated that recurrent sleep restriction affects not only spontaneous pituitary–adrenal activity but also the response to corticotropin-releasing hormone (CRH)²⁵ and to experimental stressors.^23–25 A recent human study has described an exaggerated cortisol response to a complex psychosocial stressor presented in the late afternoon following one night of total sleep deprivation.³⁰ It is not known whether this exaggerated cortisol response was the consequence of alterations in the cognitive processes involved in the stress response, and/or of alterations in other effectors of the stress response such as hypothalamic CRH activity or the pituitary–adrenal response to CRH stimulation.

The objective of the present study was to isolate the impact of sleep restriction on the pituitary–adrenal response to CRH stimulation, a downstream component of the stress response that mediates many of the physiological consequences of stress exposure. To this effect, we examined the overall ACTH and cortisol responses to an exogenous CRH injection, and estimated separately the reactivity and recovery of the pituitary–adrenal responses.³¹ We used a randomized cross-over design to compare the impact of short-term severe sleep restriction (2 nights of 4 hours in bed) versus a well-rested sleep condition (2 nights of 10 hours in bed) on the responses to a CRH injection in the early evening.

SUBJECTS AND METHODS

Participants

Volunteers responded to flyers posted around campus. Subjects were excluded by phone interview if they reported a history of endocrine or psychiatric disorders, sleep complaints, smoking, and being treated with medication. Shift work, travel across time zones in the previous 4 weeks or before the scheduled experimental sessions, and a habitual consumption of more than 2 cups of coffee or cans of caffeinated drinks were also exclusion criteria. Inclusion criteria included regular nocturnal time in bed of 7–9 hours with bedtimes between 22:00 and 24:00 and wake up times between 6:00 and 8:00, an age between 18 and 30 years, and height and weight consistent with a body mass index (BMI) below 28 kg/m². Out of the 28 subjects who signed the consent form, 9 changed their mind before starting screening procedures; we thus screened 19 subjects. The screening procedures included a physical examination and a battery of clinical tests (including a blood pressure measurement, a complete blood count, a comprehensive metabolic panel, assessment of glycated hemoglobin, and a lipid panel) that had to have normal findings. One volunteer had an abnormal lipid profile, and thus 18 subjects were enrolled. Out of these 18 subjects, 2 dropped out of the study because of a scheduling conflict, 4 were excluded because of an abnormality of their thyroid function identified only after we assayed their TSH levels, and we discontinued the participation of one subject who was non compliant with the fixed bedtimes mandated before each session. Thus, 11 healthy young non-obese men (age, median [Quartile 1; Quartile 3] 21 [21; 24] years; BMI, 24.3 [21.2; 25.3] kg/m²) completed the 2 experimental sessions of the study.

Sample size calculations were based on results obtained in a previous study of short and long sleep.⁹ In this study, we measured the rate of decline of cortisol levels from 16:00 to 21:00 to characterize the resiliency of the HPA axis. The mean difference (n = 11) between the short sleep condition and the long sleep condition was 0.133 ng/ml (1.33 µg.dl⁻¹) per hour and the standard deviation was 0.124 ng/ml. Power calculation for a design with two repeated measures indicated that a sample size of n = 11 would give a 94% power to detect the same difference (alpha = 0.05).

Experimental Protocol

The protocol was approved by the University of Chicago Institutional Review Board and all participants gave written informed consent. As shown in Figure 1, the subjects participated in 2 randomized experimental sessions conducted in randomized order in the Clinical Research Center and spaced approximately 6 weeks apart. During the week preceding each session, the subjects were asked to conform to fixed bedtimes (23:00–07:00). They were asked not to deviate from this schedule by more than 30 minutes. Naps were not allowed. Wrist activity was monitored continuously to verify compliance. Median bedtime, get up time, time in bed, and total sleep time were 23:00 [23:00; 23:24], 7:11 [7:00; 7:32], 8h 08min [8h 00min; 8h 32min], and 7h 05min [6h 38min; 7h 17min], respectively. These variables did not differ between the 2 experimental conditions (p = .13; p = .29; p = .45, and p = .20, respectively). One experimental session involved 2 nights of 10 hours in bed (22:00–08:00; well-rested condition) and the other involved 2 nights of 4 hours in bed (01:00–05:00; restricted sleep condition). Sleep was polygraphically recorded each night. During waking hours, light intensity at eye level was below 50 lux. After the second night of each sleep condition, blood samples were obtained at 20-minute intervals from 09:00 to 24:00 while meals were replaced by a constant glucose infusion at a rate of 5 g/kg/24 h and ovine CRH (1 µg/kg body weight) was injected at 18:00. CRH was injected within 30 seconds after the 18:00 sample was collected. Blood samples were obtained through a sterile heparin-lock catheter inserted in the forearm at 8:00 and kept patent with a slow drip of heparinized saline. Sampling started 1 hour after catheter insertion to avoid capturing the effect of venipuncture stress. During blood sampling, measures of perceived stress were obtained at hourly intervals using the “tense” and “calm” subscales of the Visual Analog Scales for Global Vigor and Affect³² and the five-point “nervous” subscale of the Positive Affect and Negative Affect Scale.³³

Figure 1.

Schematic representation of the experimental protocol. Subjects participated in 2 laboratory sessions presented in randomized order: one involved 2 nights of 10 hours in bed (black bars) and the other involved 2 nights of 4 hours in bed (light grey bars). Sleep was polygraphically recorded during each night. After the second night of each experimental session, bedtime was at 24:00, an injection of ovine CRH (1 µg/kg body weight) was performed at 18:00 and blood samples were obtained at 20-minute intervals from 09:00 to bedtime while meals were replaced by constant glucose infusion at a rate of 5 g/kg/24 h. Measures of perceived stress were obtained at hourly intervals. TIB: time in bed; B: Breakfast; D: dinner; CRH: corticotropin-releasing hormone; q: every.

Sleep Recording

Sleep was polygraphically recorded using a digital EEG acquisition system (Digitrace, SleepMed Inc, Columbia, SC). The recordings were scored at 30-second intervals in stages wake, N1, N2, N3, and REM (rapid eye movement) according to standard criteria.³⁴ Sleep onset and final awakening were defined, respectively, as the time corresponding to the first or last 30-second interval scored N2, N3, or REM. The following variables were determined: time in bed (ie, time interval separating lights off from lights on), sleep period (ie, time interval separating sleep onset from morning awakening), total sleep time (ie, sleep period minus duration of intrasleep wake periods), sleep efficiency (ie, total sleep time/time in bed * 100), sleep maintenance (ie, total sleep time/sleep period * 100), duration of REM sleep, duration of non-REM sleep (NREM, ie, stages N1 + N2 + N3), duration of light non-REM sleep (ie, stages N1 + N2), and duration of slow wave sleep (SWS, ie, stage N3).

Assays

Blood drawn from the indwelling catheter was collected on pre-chilled EDTA tubes for ACTH and on serum separator tubes for cortisol. EDTA tubes were immediately centrifuged at 4 ^oC. Serum separator tubes were allowed to clot at ambient temperature and were then centrifuged at 4 ^oC within 30 minutes. Plasma and serum aliquots were frozen at −20 ^oC, until assay. ACTH and cortisol were measured by immunochemiluminometric assay (Immulite, Siemens). The limit of sensitivity of the ACTH assay was 1 pg/ml and the coefficient of intra-assay variation was ≤ 9.6%. The limit of sensitivity of the cortisol assay was 1.0 µg/dL and the coefficient of intra-assay variation averaged 5%.³⁵ For each hormone, all samples from the same subject were measured in the same assay.

Analyses of ACTH and Cortisol Profiles

Post-CRH injection, significant pulses of individual ACTH, and cortisol profiles were identified and characterized using the ULTRA algorithm included in the Chronobiological Series Analyzer software (CSA; https://www.ibridgenetwork.org/#!/profiles/8055256565008/innovations/60/). A pulse was considered significant if both its increment and its decline exceeded, in relative terms, twice the intra-assay coefficient of variation. We calculated the mean basal ACTH and cortisol levels over the entire daytime period (9:00–18:00) and over the 1-hour period preceding CRH injection as multiple samples have been recommended for the assessment of the pre-challenge state.³¹ All but 2 participants displayed ACTH and cortisol responses to CRH injection that were biphasic with two significant pulses in the two sleep conditions. The individual ACTH and cortisol responses to CRH during the 6 hours and during the first and second phases of the responses were assessed by the area under the curve (AUC) above baseline pre-injection level (incremental AUC: iAUC).³¹ For both the first and second phases of ACTH and cortisol responses, we calculated the relative increment, an index of “the ability of the HPA axis to fully activate following a challenge” (reactivity), and the rate of decrease, an index of “the ability of the HPA axis to fully reset following a challenge” (recovery).³¹ Relative increments were expressed in % relative to 1-hour pre-injection levels for the first phase of response and in % relative to the preceding nadir value for the second phase of response (n = 10 for ACTH, n = 9 for cortisol). Recovery rates were calculated from the first peak to the first nadir (n = 11) and from the second peak to the second nadir (n = 10 for ACTH, n = 9 for cortisol).

Analysis of Scores of Perceived Stress

Overall mean scores for “nervous,” “tense,” and “calm” were calculated for the daytime (9:00–18:00), for the 1-hour period (17:00–18:00) preceding CRH injection and for the post-injection period (18:00–24:00).

Statistical Analysis

All group values are expressed as median [Q1; Q3]. Analyses were performed using the JMP statistical discovery software from SAS. In view of the small sample size and non-normal distribution of several output variables, non-parametric statistics were used. We compared variables obtained in the 2 laboratory sessions using the Wilcoxon test. Correlations between changes in HPA variables and changes in sleep variables during the preceding night were examined using the Spearman coefficient. Statistical significance is assumed at p values < .05 and p values between .05 and .10 are reported as trends.

RESULTS

Sleep Quantity and Quality

Table 1 summarizes the sleep variables during the 2 nights of each experimental session. The difference in total sleep time between the 2 sleep conditions averaged 5h 16min [4h 57min; 5h 26min] (10 hours in bed condition: 9h 00min [8h 45min; 9h 09min] vs. 4 hours in bed condition: 3h 48min [3h 44min; 3h 50min]; p < .001). Sleep curtailment was achieved by a 3h 30min [3h 25min; 3h 51min] decrease in the lighter stages of non-REM sleep (stages N1 + N2, p < .001) and a 1h 23min [1h 05min; 1h 46min] decrease in REM sleep (p < .001). In contrast, no significant effect was found for the time spent in deep NREM sleep, that is, SWS (p = .57). SWS appeared therefore better preserved than REM or light NREM sleep. As expected, sleep latency was reduced (−14 [−23; −8] min, p < .002) following bedtime restriction while sleep efficiency and maintenance were enhanced (+4 [2; 7] %, p < .007 and +3 [2; 5] %, p < .009).

Table 1.

Mean Sleep Characteristics of the Two Nights of the 2 Sleep Conditions.

			10 h in bed	4 h in bed	p
Total sleep time (min)			540 [525; 549]	228 [224; 230]	.001
Sleep latency (min)			23 [17; 34]	10 [7; 11]	.002
Sleep efficiency (%)			89 [87; 91]	95 [93; 95]	.007
Sleep maintenance (%)			95 [94; 97]	99 [98; 99]	.009
Time spent in sleep stages (min)
WASO			31 [19; 36]	3 [2; 4]	.001
Light NREM sleep			321 [290; 351]	117 [75; 145]	.001
SWS			78 [54; 113]	63 [38; 120]	.57
REM sleep			130 [111; 150]	44 [38; 49]	.001

Light NREM = lighter stages of non REM sleep (ie, stages N1 + N2); REM = rapid eye movement; SWS = deepest stage of non REM sleep (ie, stage N3); WASO = wake after sleep onset. p values are from Wilcoxon exact rank test. Data are expressed as median [Quartile 1; Quartile 3].

ACTH and Cortisol Profiles Before and After CRH Injection

Figure 2a shows the ACTH and cortisol profiles observed in the 10 hours in bed and in the 4 hours in bed conditions. Prior to CRH injection, relative to the well-rested condition, sleep restriction was associated with a 36% increase in daytime ACTH levels (mean 9:00–18:00: +36 [+4; +53] %; p = .032) without significant increase in daytime cortisol (mean 9:00–18:00: +9 [−12; +29] %; p = .58; Figure 2b).

Figure 2.

(a). ACTH and cortisol profiles pre- and post- CRH injection at 18:00 in the 10 hours in bed condition (left panels) and in the 4 hours in bed condition (right panels). The solid lines represent the median and the dotted lines represent the 25th and 75th percentiles. The time of CRH injection is indicated by the arrows. (b). Box and whisker plots of means of ACTH and cortisol pre-oCRH injection, that is, from 9:00 to 18:00. The line inside the box shows the median, the ends of the box are the 25th and 75th percentiles, the whiskers represent the largest and smallest observed values that are not outliers, and the dots represents outliers defined as 1.5 * interquartile range. p levels are from Wilcoxon test. ACTH: adrenocorticotropic hormone, CRH: corticotropin-releasing hormone.

As previously reported in studies where the post-CRH sampling period extended beyond the typical 90–120 minutes of clinical testing,^36,37 pulse analysis revealed biphasic ACTH and cortisol responses to CRH injection for most of the subjects (10 out of the 11 subjects for ACTH and 9 out of the 11 subjects for cortisol) (Figure 2a and 3a). For a given subject, the qualitative characteristics of the ACTH and cortisol responses were similar for both sleep conditions. The first peak of ACTH occurred 40 minutes after CRH injection in both sleep conditions (p = .21) and was followed by a second pulse that peaked, on average, 2 to 3 hours after CRH injection, irrespective of the sleep condition (p = .28). As expected, cortisol pulses followed the ACTH pulses by roughly one sampling interval, that is, 20 minutes. Irrespective of the sleep condition, the first pulse of cortisol was detected 1 hour after CRH injection (p = 1) and the second pulse occurred 3 hours later (p = 1). For both ACTH and cortisol, and for both sleep conditions, the peak value of the second pulse was lower than the peak value of the first pulse (p < .009 for all). Compared to the rested condition, the overall response was reduced by 27 [17; 28] % (p = .002) for ACTH and by 21 [8; 46] % (p = .083) for cortisol after sleep restriction (Figure 3b).

Figure 3.

(a). Relative ACTH and cortisol responses to CRH injection in the 10 hours in bed condition (left panel) and in the 4 hours in bed condition (right panel). The solid lines represent the median and the dotted lines represent the 25th and 75th percentiles. (b). Box and whisker plots of overall ACTH and cortisol responses to CRH injection (AUC above baseline 18:00 to 24:00). The line inside the box shows the median, the ends of the box are the 25th and 75th percentiles, the whiskers represent the largest and smallest observed values that are not outliers, and the dots represents outliers defined as 1.5 * interquartile range. p levels are from Wilcoxon test. ACTH: adrenocorticotropic hormone, CRH: corticotropin-releasing hormone, AUC: area under the curve.

Summary values for the iAUC, relative increment and rate of recovery of the first and second phases of ACTH and cortisol responses are presented in Table 2. The overall response (iAUC), the relative increment and the rate of recovery of the first phase of the cortisol response were decreased during sleep restriction (−14 [−34; −8]%; p = .019, −21 [−29; −16] %; p = .002, and −21 [−31; 0] %; p = .032, respectively; Table 2), without significant difference between sleep conditions for ACTH. No differences between sleep conditions were observed for the second phase of the response for either of these hormones.

Table 2.

Characteristics of the First and Second Phase of the Adrenocorticotropic Hormone (ACTH) and Cortisol Responses to Corticotropin-Releasing Hormone (CRH) Injection at 18:00.

	ACTH			Cortisol
	10 h in bed	4 h in bed	p	10 h in bed	4 h in bed	p
First phase of response to CRH
AUC above baseline	1.7 [1.5; 2.2]	1.7 [1.3; 2.3]	.37	1.3 [0.9; 1.4]	0.9 [0.8; 1.2]	.019
Relative increment (%)	296 [266; 544]	240 [210; 550]	.21	211 [166; 282]	147 [131; 195]	.002
Recovery rate	25.0 [11.4; 28.0]	18.8 [11.9; 27.5]	.97	9.3 [5.1; 12.2]	6.5 [4.8; 8.3]	.032
Second phase of response to CRH
AUC above baseline	0.8 [0.5; 1.2]	0.6 [0.3; 0.8]	.13	1.2 [1.0; 1.2]	0.6 [0.3; 1.5]	.43
Relative increment (%)	62 [36; 109]	69 [47; 83]	.70	63 [41; 67]	48 [36; 64]	.73
Recovery rate	4.0 [3.8; 4.9]	2.8 [2.6; 4.9]	.49	2.9 [2.7; 5.1]	3.2 [2.8; 3.8]	.91

ACTH = adrenocorticotropic hormone; AUC = area under the curve; CRH = corticotropin-releasing hormone. Data are expressed as median [Quartile 1; Quartile 3]. p levels are from Wilcoxon test. The ACTH and cortisol responses to CRH injection were biphasic for most of the subjects. The AUC above baseline, the relative increment and the recovery rate of the 2 phases of the response are presented. AUCs above baseline depict the overall responses that include both the ascending and the descending portion, defined as the portions of the curve from the nadir to the peak and from the peak to the nadir, respectively, of each significant pulse identified by the ULTRA algorithm. AUCs are expressed in 10³pg.ml⁻¹.min for ACTH and 10³µg.dl⁻¹.min for cortisol. Relative increments capture the magnitude of the ascending phase, ie, an index of the ability of the HPA axis to fully activate. They are expressed in % relative to 1h pre-injection levels for the first phase of response (n = 11) and in % relative to the preceding nadir value for the second phase of response (n = 10 for ACTH, n = 9 for cortisol). Recovery rates represent an index of the ability of the HPA axis to reset following a challenge. They are expressed in pg.ml⁻¹.h⁻¹ for ACTH and in µg.dl⁻¹.h⁻¹ for cortisol and were calculated from the first peak value to the first nadir value (n = 11) and from the second peak value to the second nadir or minimal value reached towards the end of the test (n = 10 for ACTH, n = 9 for cortisol). Bold text indicates a statistically significant difference with a p-value less than .05.

Correlations Between Sleep Variables and Pituitary–Adrenal Responses

Correlation analyses were conducted to explore whether individual differences in the changes in ACTH and cortisol responses were partly predicted by individual differences in total sleep time achieved during each experimental condition. The reduction in total sleep time from the 10- to 4-hour bedtime condition ranged from 4h 42min to 5h 38min. This reduction was negatively correlated to the changes in overall response (18:00–24:00 iAUC) of ACTH and cortisol such that individuals who experienced larger amounts of sleep loss also had greater reductions in overall ACTH and cortisol responses to CRH injection (r_Sp = −0.65, p = .032, Figure 4a and r_Sp = −0.71, p = .015; Figure 4b). We performed a sensitivity analysis to determine whether these negative correlations were driven by one of the 11 subjects, removing one subject at a time and repeating the calculations with n = 10. In the case of cortisol, irrespective of the subject removed, the correlation remained significant with only one exception where the p level became .060. In the case of ACTH, significance or near significance (p = .060) remained in 8 of 11 analyses with n = 10. No significant correlations between the difference in total sleep time and differences in ACTH/cortisol reactivity and recovery between the two sleep conditions were detected.

Figure 4.

Associations between the reduction in total sleep time (assessed by subtracting the total sleep time on the last 4 hours night to the total sleep time on the last 10 hours night) and the changes in pituitary–adrenocortical response to CRH injection (AUC above baseline 18:00 to 24:00). The reduction in total sleep time was inversely correlated to the changes in ACTH (a) and cortisol (b) overall response after CRH injection. ACTH: adrenocorticotropic hormone, CRH: corticotropin-releasing hormone.

Measures of Perceived Stress

The mean profiles of hourly scores of self-perceived stress for both the well-rested and the sleep restricted conditions are shown in Figure 5 and reveal similar low levels of perceived stress irrespective of bedtime condition. The overall mean scores of perceived stress for both the entire daytime pre-injection and the 1-hour pre-injection periods were similar in the two sleep conditions (9:00–18:00: “nervous”: p = .25; “tense”: p = .21, and “calm”: p = .17; 17:00–18:00: “nervous”: p = .10; “tense”: p = .83, and “calm”: p = .98). Similar results were obtained for the post-injection period (“nervous”: p = .50; “tense”: p = .97 and, “calm”: p = .70, respectively).

Figure 5.

Hourly scores on the five-point scale for “nervous” and on the 10-cm visual analog scales for “tense” and “calm” after 2 nights of 10 hours in bed (left panels) and after 2 nights of 4 hours in bed (right panels). The solid lines represent the median and the dotted lines represent the 25th and 75th percentiles. The arrows represent the time of CRH injection (18:00). There were no significant effects of time in bed duration on any of these three markers of self-perceived stress measures for both the pre- and post- CRH injection periods. CRH: corticotropin-releasing hormone.

DISCUSSION

The present study examined the effects of restricted sleep, as compared to a well-rested condition, on the response of the pituitary–adrenal axis to a standard CRH stimulation test in healthy subjects under controlled laboratory conditions. Two nights of 4 hours in bed were associated with a 27% reduction in ACTH response to CRH injection, suggestive of a decreased pituitary sensitivity to CRH in a state of sleep debt. A non-significant trend for a similar reduction (21%) in the overall cortisol response was also observed. Furthermore, the rapid elevation of cortisol levels in response to CRH injection was 21% lower when the participants were in a state of sleep debt while no significant change in the acute ACTH response was detected. Lastly, restricted sleep was associated with reduced resilience of the adrenal response axis, as the rate of decrease of cortisol levels after CRH injection was decreased by 21%, suggesting impaired glucocorticoid recovery. The time course of the pituitary response was not affected. In sum, these findings indicate that the impact of insufficient sleep on HPA axis activity includes not only the increase in basal ACTH and glucocorticoid levels previously reported in studies of severe sleep restriction,^8–17 but also a blunting of the pituitary–adrenal response to CRH, a pivotal element of the neuroendocrine response to most, if not all, stressors.

There have been very few experimental studies examining alterations of HPA function following sleep deprivation. Our results are in line with a study in rats that reported a blunted ACTH response to CRH injection after recurrent partial sleep loss.²⁵ The decrease in humans and rodents ACTH release after CRH injection is consistent with a reduction of pituitary sensitivity to CRH in a state of sleep debt, a hypothesis supported by the decreased CRH binding found in pituitary glands of sleep-deprived rodents.³⁸ Findings of elevated expression and release of CRH in sleep-deprived rodents^38–40 and of elevated baseline ACTH levels in sleep-deprived humans in the present and in a previous study¹⁶ suggest that partial pituitary desensitization to CRH is likely the consequence of sustained elevated CRH release. Elevated CRH release, a marker of physiologic arousal, could be conceptualized as an adaptive mechanism designed to stimulate alertness in a condition where sleepiness and sleep propensity are increased.

Whether these results may be transposed to social, emotional, or biological stressors remains, however, an open question. A limited number of rodent studies that have observed a blunted ACTH response to a stress challenge in the face of unchanged corticosterone response^23–25 are in favor of this hypothesis. However, sleep loss in humans has been shown to intensify the negative emotional perception of disruptive and unforeseen events⁴¹ and to lower the threshold at which a cognitive demand is perceived as stressful.⁴² These observations suggest that sleep loss induces an alteration in the perception of environmental challenges on a cognitive level that is likely to be associated with changes in the afferent inputs to neuroendocrine regions. Thus, under real life conditions, sleep restriction may involve an increased response to the multiple, even modest, daily life stressors rather than a blunting of pituitary–adrenal responses as observed in our laboratory study designed to isolate the response to exogenous CRH injection. Supporting this alternative hypothesis, a recent study has described an exaggerated cortisol response to a complex psychosocial stressor presented in the late afternoon following one night of total sleep deprivation.³⁰ More research is clearly needed to examine the impact of insufficient sleep on stress-responsive systems.

It has been hypothesized that the elevation of evening cortisol levels evidenced after insufficient sleep^8–14,16 reflects altered HPA axis recovery from the circadian-driven morning stimulation.^8–10 The present study supports this hypothesis since we observed a 21% reduction in the rate of decrease of cortisol levels after CRH injection after just 2 nights of restricted sleep compared to a well-rested condition. Reduced resilience of the HPA axis seems therefore to characterize both the spontaneous and stimulated states after sleep restriction.

Our study has limitations. First, the sample size is small, which precludes the detection of small or medium-sized effects.⁴³ However, this limitation was partly mitigated by our a priori sample size calculation and by the highly controlled experimental conditions and the in-depth phenotyping of the pituitary–adrenal response. Second, we studied only young men and the data cannot, therefore, be extrapolated to the general population. Third, we examined the response to CRH only at one time of day, coinciding with the beginning of the quiescent period of HPA activity. Since adrenal sensitivity appears to be differently affected in the morning and in the evening during sleep loss,¹⁶ the response to CRH administered in the morning may differ from that observed in the evening. Fourth, sleep was severely restricted but only for 2 days while under real life conditions insufficient sleep is often a chronic condition involving a daily sleep loss of one to two hours. Lastly, although necessary to ensure robustness and reliability of our results, our well-controlled design (including continuous glucose infusion to avoid the impact of timing and content of meals on cortisol secretion^44,45 and light levels below 50 lux) does not mimic real life and cannot, therefore, be generalized. This study also has several strengths. First, we used a randomized cross-over design with rigorously controlled experimental conditions. Second, we assessed both ACTH and cortisol by frequent sampling for 6 hours post-CRH injection, therefore allowing for a complete assessment of pituitary–adrenal response. Lastly, we showed that perceived stress, assessed at frequent intervals during the entire study, was not affected by either sleep restriction or intravenous CRH injection, suggesting that we were able to isolate the response to CRH from potential effects of associated psychological stressors.

In conclusion, we found that 2 nights of sleep restriction result in significant alterations of HPA functioning, including enhanced spontaneous activity and blunted response and reduced recovery to an exogenous challenge. These alterations are likely to be the consequence of sustained elevation in CRH release, reduced pituitary sensitivity to CRH and impaired glucocorticoid recovery. Irrespective of the underlying mechanisms, these abnormalities in HPA functioning may mediate the association between insufficient sleep and increased risk for various conditions, including obesity, diabetes, cardiovascular diseases, vulnerability to infection, depression and anxiety.

FUNDING

The work described in this article was supported by US National Institute of Health grants DK-41814, HL-075025, P01 AG-11412, P60 DK-20595, and ULl-TR000430 to the University of Chicago and HL-075025 to the Université Libre de Bruxelles, by a grant from the European Sleep Research Society (ESRS) and by the WAKING team, Lyon Neuroscience Research Center, INSERM U1028-CNRS UMR 5292, Claude Bernard University, Lyon, France. Aurore Guyon was supported by a fellowship from the French Society for Research & Sleep Medicine (SFRMS) (France). The funding sources had no role in the design, conduct, or reporting of this study.

DISCLOSURE STATEMENT

AG, LM, MB, ET, RL, and MLB have nothing to declare. EVC receives grant support from Astra Zeneca, Merck, and Shire Inc., and is a consultant for Philips/Respironics, Shire Inc., Pfizer Inc. and Vanda. KS received Speaker’s honorarium from Novo Nordisk.