jet lag and cholesterol synthesis

Diurnal rhythmicity of human cholesterol synthesis: normal pattern and adaptation to simulated “jet lag”

LYNN KESSLER CELLA, EVE VAN CAUTER, AND DALE A. SCHOELLER Committee for Human Nutrition and Nutritional Biology and Department of Medicine, University of Chicago, Chicago, Illinois 60637

Cella, Lynn Kessler, Eve Van Cauter, and Dale A. Schoeller. Diurnal rhythmicity of human cholesterol synthesis: normal pattern and adaptation to simulated “jet lag.” Am. J. Physiol. 269 (Endocrinol. Metab. 32): E489-E498, 1995x- The diurnal rhythm of cholesterol synthesis was determined by deuterium incorporation from body water in five normolipe- mic men studied during a 24-h baseline period and on the lst, Znd, and 4th days of a simulated 12-h time zone shift achieved by delaying sleep times and, starting on the 2nd day, mealtimes. Profiles of plasma cortisol and thyrotropin (TSH) were obtained simultaneously. Under baseline conditions, cholesterol synthetic rates varied from essentially zero in the morning to maximal values around midnight. On the 1st shifted day, this diurnal variation was unaltered despite sleep-wake reversal. The diurnal pattern of cholesterol synthesis, however, was shifted 5 h on the 2nd shifted day and N 12 h on the 4th. The diurnal variation of synthetic rate cholesterol fractional synthesis and plasma cortisol levels was negatively correlated on both the baseline day and the 1st shifted day. A positive correlation with the TSH rhythm was found on the 1st day only. During the 2nd and 4th days, the rhythm of cholesterol synthesis adapted faster than the rhythms of cortisol and TSH. These findings indicate that cholesterol synthesis is not acutely entrained by the sleep-wake cycle nor is it primarily entrained by the circadian clock.

deuterium; mass sol; thyrotropin

spectroscopy; circadian rhythm; sleep; corti-

Maximal synthetic rates were found to occur in the early morning hours or N 2-6 h later than estimated from the above investigation of precursor levels. However, in a subsequent study by Jones et al. (15), maximum cholesterol synthesis occurred near that reported for the above-mentioned precursor studies. Moreover, it was found that profiles of the diurnal variation in cholesterol synthesis as measured by deuterium incorporation coin- cided with those of the plasma mevalonate level method. The lack of standardization of sleep times, light-dark exposure, and mealtimes and the relatively infrequent sampling intervals could account for the discrepancies in time of maximal synthesis observed in these studies.

A diurnal rhythmicity of cholesterol synthesis has also been clearly established in experimental animals. A diurnal variation in synthesis has been demonstrated in the liver, and, with a somewhat lesser amplitude, in the intestinal mucosa (8). In nocturnal animals fed ad libitum, the rate of cholesterol synthesis is highest after midnight during the dark cycle and lowest during the morning and early afternoon (8). This rhythmic variation has been associated with changes in the activity of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, the rate-limiting enzyme in cholesterol synthesis (l&25). In the rat, the diurnal rhythm of cholesterol synthesis appears to be intimately related to food intake. A shift in feeding sched ule without changi .ng the lighting cycl .e is followed by a shift in the rhythm that stays in phase with feeding (8). However, whereas intestinal cholesterol synthesis shifts rapidly to stay in phase with feeding (8, 9), hepatic synthesis does not come com- pletely into phase when feeding times are changed (8), suggesting that other factors, such as intrinsic circadian rhythmicity, may also play a role. Furthermore, adrenalectomy has been shown to decrease (19) or abolish (7) the nocturnal rise in HMG-CoA reductase activity, suggesting a role for corticosterone in the regulation of the daily rhythm of this enzymatic activity. More recently, Waurin and Schibler (31) have demonstrated a circadian rhythm in an HMG-CoA reductase transcrip- tion activator protein DBP, which free-runs under constant conditions, independently of food intake, and could be related to diurnal changes in cholesterol synthesis. Thus, at least in laboratory animals, evidence exists that both feeding schedule and circadian rhythm play a role in regulating the diurnal variation of cholesterol synthesis.

IT IS WELL ESTABLISHED that de novo cholesterol synthesis is a major source of cholesterol entering the body pool in humans. Moreover, it has been demonstrated that this rate of synthesis varies dramatically across the day (14,17,18,22). Few studies, however, have investigated the factors that regulate this diurnal variation because of a lack of methods for measuring within-day changes in cholesterol synthesis in vivo. This is unfortunate because investigations of cholesterol metabolism in humans during sleep deprivation (30) and shift work rotations (26) suggest that changes in sleep-wake and/or light-dark cycles may alter cholesterol homeostasis.

Among those who have investigated the diurnal variation of cholesterol synthesis, Miettinen (18) observed a nocturnal increase in the plasma levels of the cholesterol precursors squalene and lanosterol, with maximal levels between midnight and 0400. In addition, Parker et al. (22) observed a nocturnal increase in plasma levels of the precursor mevalonate, consistent with an overall augmentation in the biosynthetic pathway. Direct mea- sures of diurnal variations in human cholesterol synthesis have only been recently achieved using the deuterium incorporation technique. In one study (17) measurements of cholesterol synthesis were obtained at 4-h intervals over a 48-h period in six young men.

In view of the paucity of data regarding diurnal variations in human cholesterol synthesis and of the uncertainties regarding their possible control mecha- nisms, the aim of the present study was to characterize 24-h changes in human cholesterol synthesis under controlled feeding, lighting, and sleeping conditions, and

0193-1849/95 $3.00 Copyright o 1995 the American Physiological Society E489

E490 DIURNAL RHYTHM OF HUMAN CHOLESTEROL SYNTHESIS

Table 1. Physical parameters and lipid concentrations Experimental Protocol

Screening Plasma Levels, Prestudy habituation. The study was conducted at The mmol/l

Age, Height, Weight, Body University of Chicago. For 1 wk before the study, all the

Subjects yr cm kg Fat, % Cholesterol Triglycerides subjects carried out their usual work and social activities but were asked to adhere to a standardized schedule of sleep (from

Treatment group

1 23 170 73.0 20 5.15 1.40 2 23 182 92.0 19 3.93 1.00 3 22 184 65.4 17 2.72 0.55 4 25 183 70.1 8 2.87 0.82 5 24 171 87.6 20 4.60 0.61

Means IL SD 23 2 1 178+ 7 77.6 2 11.5 17 + 5 3.85 + 1.06 0.88 +000.34

Control group

6 25 192 93.0 25 5.07 1.31 7 24 182 81.8 20 3.96 0.52

2300-0700) and meals (at 0730,1230, and 1730) to maximize interindividual synchronization and fully establish baseline conditions. During this period the subjects were asked to abstain from alcohol or caffeine consumption. For 3 days before the study period the subjects were provided with standardized breakfast, lunch, and dinner meals composed of Western-style foods, which the subjects consumed in the Clinical Research Center (CRC) or were allowed to take with them. Although the time to stabilize the diurnal rhythm of cholesterol synthesis had not been investigated before our current study, 3 days was selected because previous studies had demonstrated that unlike plasma cholesterol level, which

to test whether they are entrained to a sleep or the circadian clock. The rate of adaptation of the temporal pattern of cholesterol synthesis was compared with that of simultaneously measured 24-h profiles of cortisol, a hormone that is markedly influenced by circadian rhythmicity, and thyrotropin (TSH), a hormone that is markedly influenced by circadian rhythmicity and sleep (29).

METHODS

is slow to respond to a change in diet, the diurnal variation of cholesterol fractional synthetic rate (FSR) responds very rapidly to intervention (14, 16). Meal composition was 40% carbohydrate, 25% protein, 35% fat with 170 mg cholesterol/ 1,000 calories, and a polyunsaturated-saturated fat ratio of 0.8. Meals were prepared in the CRC metabolic kitchen. Energy was provided to maintain weight as calculated from a measured postabsorption resting metabolic rate, using a Delta- trac metabolic cart (Sensormedic, Yorba Linda, CA), multi- plied by 1.1 for dietary-induced thermogenesis and 1.65 (outpa-

Subjects tient) or 1.4 (inpatient) activity factor to meet daily energy needs.

Volunteers from the university community provided a medi- BaseLine day. The subjects spent the night before the study cal history, and seven subjects were selected who reported that in the CRC to habituate to inpatient sleeping conditions, i. e., they were healthy nonsmokers without cardiovascular, liver, with a forearm venous catheter and polygraphic sleep record- renal, endocrine, lipid, or psychiatric disorders and were ing electrodes in place. After this habituation night the taking no medication (Table 1). Shift workers or subjects who subjects were released from the CRC but fed the habituation had experienced a transmeridian flight within 6 wk before the diet. Subjects were readmitted to the CRC on the same day at study were excluded. Positive factors for selection included 1600 and remained there for the next 5 days (Fig. 1). A regularity of work, social, meal, and sleep schedules. Fasting forearm venous catheter for blood sampling was inserted and lipid levels and blood glucose were within normal ranges (total kept patent with a saline drip (75 ml/h). Dinner was served at cholesterol 2.59-5.20 mmol/l, fasting triglyceride 0.1-1.8 1730. During the sleep periods the catheter was connected to a mmol/l, fasting glucose 3.9-6.1 mmol/l) in all individuals plastic tube extending to the adjacent room so that sampling studied. Diet histories indicated a habitual consumption of could continue without disturbing the subject. A baseline 34 t 4% (SD) of daily energy intake from fat and a cholesterol blood sample was obtained at 1800, after which a priming dose consumption of 187 t 81 (SD) mg/l,OOO calories. The nature of deuterium oxide (1.0 g 2H20/kg estimated body water, 99.8 and intent of the study were explained, and written informed atom% excess, tritium depleted, MSD Isotopes, St. Louis, MO) consent was obtained. All procedures were approved by the was orally administered. Blood samples were collected hourly Institutional Review Board at The University of Chicago for the next 72 h. Deuterated oral fluids were provided Medical Center. throughout the study to maintain constant plasma water

& Loading dose D20 lg/kgTBW )-- Deuterated drinking water I

I- I.V. saline drip I I 4 )-- Hourly blood sampling --I I i

Fig. 1. Schematic representation of the 1-1 Sleep recording 1-1 I I 5-day simulated jet-lag protocol. Com- plete darkness is indicated by the solid horizontal bar, simulated daylight by the open bar, and dim by the hatched bar. Meals are indicated as breakfast

Day 2 Day 3 Day 4 Baseline I

Day 1 I I I I

(B), lunch (Id, and dinner (D). DZO, I

2Hz0 or deuterium oxide; TBW, total body water; IV, intravenous. 1800 1800 1800 1800 1800 1800

Meals BLD BL B LD BLD BLD

DIURNAL RHYTHM OF HUMAN CHOLESTEROL SYNTHESIS E491

enrichment. Because oral fluids only comprise about one- fourth of estimated water input, all oral fluids contained 4.2 g 2H20/kg total body water. The subjects slept in total darkness from 2300-0700. At 0700 the subjects were awakened and the room was illuminated with conventional indoor and natural outdoor lighting. Breakfast was served at 0730, lunch at 1230, and dinner at 1730. The subjects were allowed 30 min to consume their meals and were not allowed any other foods or fluids. During waking hours the subjects were allowed to walk around the unit, watch television, listen to the radio, and engage in conversation with visitors and staff personnel. Naps were strictly prevented. Two subjects stayed on this schedule of bedtimes, mealtimes, and light exposure to examine the stability and reproducibility of metabolic and hormonal profiles during prolonged (i.e., 4 days) hospitalization in the CRC. The other five underwent a simulated jet lab as described below.

Day 1, sleep deprivation- 12-h simulated time zone shift. At 2300 the overhead lights in the room were dimmed but the subjects were not allowed to sleep. At 0700 overhead lights were turned on and natural outdoor light was allowed into the room. Breakfast was served at 0730 (i.e., as on the baseline day) and lunch at 1030 (i.e., 2 h earlier than on the baseline day) while the subjects were kept continually awake. At 1100, i.e., 12 h later than during baseline conditions, blackout drapes were drawn and the subjects were allowed to sleep in total darkness. At 1800 hourly blood samples had been obtained continuously for 48 h and day 2 began.

Days 2-4 of the 12-h shift. The subjects were awakened at 1900, i.e., 12 h later than under baseline conditions. A mobile panel of fluorescent tubes providing a light intensity of 5,000~5,500 lux at 2 ft were turned on and placed 4 ft from the subject to simulate daylight (Sunbox, Rockville, MD). Subjects received breakfast at 1930, lunch at 0030, and dinner at 0530, i.e., 12 h later than under baseline conditions. At 0700 the light panels were turned off and overhead lights in the room were dimmed to simulate evening. Subjects slept in total darkness from 1100 to 1900. Blood sampling was terminated at 1800, i.e., after 72 h of hourly collections. The subjects were maintained on this 12-h shift schedule for an additional 48 h, i.e., days 3 and 4. Blood sampling at hourly intervals was reinitiated at 1800 on day 3 and continued for the final 24 h of the study.

Laboratory Procedures

De novo choZesteroZ synthesis. De novo cholesterol synthesis was measured by the deuterium incorporation method (16). Deuterium enrichment was measured in plasma total cholesterol and plasma water. Neutral lipids were extracted from 1 ml plasma samples (4). Samples were mixed with 10 ml chloroform-methanol 2: 1 (vol/vol) and filtered. The filtrate was back-extracted with 2.5 ml of 0.1 mol/l KC1 and the upper phase discarded. The lower phase was washed three times with 5.0 ml chloroform-methanol-O.1 mol/l KC1 3:48:47 (vol/vol/ vol). Solvent was evaporated at room temperature in a vortex evaporator. Extracts were saponified at 50°C in 0.4 mol/l ethanolic potassium hydroxide overnight. After adding 1 ml H20, the nonsaponifiable fraction containing free cholesterol and deesterified cholesterol was extracted three times with 2 ml petroleum ether, which was back-extracted with 4.0 ml of 0.1 mol/l ethanolic potassium hydroxide. Solvent was evaporated at room temperature in a vortex evaporator. The isolated cholesterol was transferred to a 6-mm-OD quartz tube using three washes of chloroform. Solvent was evaporated, and 0.1 g cupric oxide and a 2-cm length of silver wire were added before flame sealing under vacuum. Samples were cornbusted to CO2 and H20 at 750°C for 2 h. The Hz0 was isolated via vacuum

distillation, cryogenically transferred to a second quartz tube, and reduced to H2 over 0.05 g zinc reagent (Friends of Geology, Bloomington, IN) at 500°C for 30 min. The resulting H2 was then analyzed by gas isotope ratio mass spectrometry using a triple inlet Nuclide 3-60 HD-RMS (PATCO, Belefonte, PA) (16). Abundances were expressed as parts per million (ppm) deuterium (mol/mol) based on the international Standard Mean Ocean Water (155.76 ppm). Isotope abundances were corrected for exchange during combustion-reduction (unpublished data). Exchange averaged 2.4 I_LM Hz with deuterium abundance of 148 ppm. To measure deuterium enrichment of body water, postdeuterium plasma samples were gravimetri- tally diluted lo-fold with distilled water to reduce the deuterium enrichment to within the normal analytic range. Baseline samples were not diluted. Triplicate 2-l_~1 samples were vacuum distilled, reduced, and mass spectrometrically analyzed as previously described.

Cortisol assay. Hourly plasma cortisol levels were measured by radioimmunoassay (Diagnostic Products, Los Angeles, CA) with an average intra-assay coefficient of variation of 4.0%.

TSH assay. Hourly TSH levels were measured by radioimmunoassay (Serono Diagnostics, Coinsins, Switzerland) with an average intra-assay coefficient of variation of 5.8%.

Plasma lipid ZeveZs. Total and esterified plasma cholesterol levels were measured enzymatically (Lancer’s Kits, Division of Sherwood Medical, St. Louis, MO). Free plasma cholesterol levels were not directly measured but determined by difference from total and esterified cholesterol. Triglyceride levels were measured enzymatically (Boehringer-Mannheim, Indianapo- lis, IN).

Cakulation of CholesteroZ FSR

FSR was calculated for each 2-h interval from plasma total cholesterol deuterium enrichment and plotted at the midpoint of the time interval. In previous studies, plasma free cholesterol had been used to measure de novo cholesterol synthesis (16) because of the observation that newly synthesized cholesterol is released into the circulation as free cholesterol (24). However, we have recently observed that the appearance of deuterium in free cholesterol demonstrates a substantial nonlinearity after the first 24 h (unpublished data). This results in an underestimation of FSR during subsequent time periods. The sampling of total cholesterol substantially re- duces this problem. FSR was calculated assuming labeled hydrogen incorporation per carbon atom (H/C ratio) of 0.81 (6), i.e., 22 deuterium atoms (D) out of 46 total hydrogen atoms (H) in the 27 carbon (C) molecules

FSR (pools/day)

delta ppm (cholesterol) x 12

= excess ppm (plasma water) x 22D/27C x 27C/46H

where delta ppm (cholesterol) is the difference in the deuterium enrichment across the 2-h sampling interval and excess ppm (plasma water) is the average deuterium enrichment of plasma water above baseline during the sampling interval. The factor of 12 converts the 2-h FSR value to a 24-h rate.

Sleep Recording and Analysis

The polygraphic sleep records were scored visually at 30-s intervals in stages wake, I, II, III, IV, and rapid eye movement (REM) according to the standardized criteria (23). Sleep onset and morning awakening were defined, respectively, as the times of occurrence of the first and last 30-s intervals scored II, III, IV, or REM. The sleep period was defined as the interval separating sleep onset from final morning awakening. Sleep


efficiency was calculated as total time in bed minus total duration of wake divided by the total time in bed.

Statistical Analysis

A 1:2:1 weighed 3-point moving average was used to calcu- late a smoothed curve for each individual FSR profile. The acrophase (maximal FSR), nadir (minimal FSR), and amplitude (50% of the difference between the maximal and minimal rate expressed as a percent of the 24-h mean level) were identified on the individual smoothed curves within each 24-h interval (baseline, day 1, day 2, and day 4). Reported mean values were calculated from the individual values and not from the mean levels plotted in Figures 3-8. For cortisol and TSH profiles, a best-fit curve quantifying the circadian variation was obtained using a robust weighted regression procedure (5). The best-fit curve was obtained using the robust, locally weighed, regression technique described by Cleveland (5) with a window of 8 h, i.e., t4 h. This procedure involves smoothing the data within a moving 8-h window by weighted least-square polynomial fit, with the weights being largest at the center of the window and smallest at the limits of the window. The acrophases, nadirs, and amplitudes were calculated from the best-fit curve as for the individual FSR profiles. The effect of study day on the acrophase, nadir, and amplitude of the FSR and hormonal profiles and on the 24-h mean FSR and hormonal levels was determined using analyses of variance (ANOVA) for repeated-measure analysis and a post hoc paired t-test with a Bonferroni correction.

The relationship between cholesterol FSR and hormone levels was quantified by calculating the coefficient of cross correlation (13) for the individual cholesterol FSR and cortisol- TSH profiles for each 24-h interval (baseline, day 1, day 2, and day 4) at time lags of 0 h (i.e., simultaneous values of cholesterol FSR and cortisol-TSH), of: 1 h (i.e., cholesterol FSR leadingcortisol-TSH by 1 h or visaversa), 22, +3, +4, 25, and t6 h. Each cholesterol FSR profile was determined from 12 measured and 12 interpolated values. For each pair of profiles, the largest coefficient of cross correlation was identified.

Time effects on plasma cholesterol, plasma triglyceride, and plasma water deuterium enrichment were analyzed by regress- ing plasma concentrations against time. Means t SE are presented unless otherwise stated.

RESULTS

Plasma Lipids, Body Weight, and Precursor Enrichment

No significant time effects were observed on plasma- free cholesterol over the study period, but ANOVA revealed that plasma total cholesterol levels differed with respect to study day (Fig. 2; P = 0.04). Post hoc analysis indicated that baseline levels were 0.2 to 0.3 mmol/l lower than those of the other 3 days (P < 0.05). No significant changes in fasting plasma triglyceride levels were observed over the study period. Mean body weight was stable within 0.8%, with a mean change of 0.2 t 0.9 kg [not significant (NS)]. The grand mean of the plasma water deuterium enrichments was 841 t 11 ppm (Fig. 3). The within-subject coefficient of variation in enrichment averaged 2.0%. No significant time effects were observed on mean plasma water deuterium enrichment over the study period.

6

3.00

- 1

a r 2.00

6 s 1.00

t - -

o~~~*~~~p-p-p-p-o~~-o-o-o------- - - -o- o- o- 0 0

0.00 ’ ’ ’ ’ ’ ’ ’ ’ 1 ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ 1 1800 1800 1800 1800 1800 1800

BL Dl D2 D3 04 DS

TIME (time/day)

Fig. 2. Plasma free (--o--) and total (-•--> cholesterol levels for the 5 simulated jet-lag subjects; means + SE. Bl, baseline; Dl-D5, days l-5.

Cholesterol FSR, Cortisol, and TSH Across the Study

ANOVA indicated that 24-h cholesterol FSR was stable across the study period, averaging 0.042 t O.Oll/ day on the baseline day, 0.051 t O.O07/day on shifted day 1, 0.047 t O.Oll/day on day 2, and 0.035 t O.OOS/day on day 4 (NS). The 24-h mean cortisol level was 157 t 11 nmol/l on the baseline day, 184 t 11 nmol/l on day 1,193 - + 11 nmol/l onday2, and 204 t 17 nmol/l on day 4, but the trend was not significant with time (P = 0.17). The 24-h mean TSH level increased with time (P = 0.01) from 1.9 t 0.4 mU/l on the baseline day to 2.7 - + 0.5 mu/l on day 1, 2.3 t 0.3 mu/l on day 2, and 2.9 t 0.5 mu/l on day 4, although only day I and day 4 differed by post hoc t-test (P < 0.05).

Reproducibility of Baseline Profile of Cholesterol FSR

The profiles obtained in the two subjects who were studied without shift of sleep times, mealtimes, and exposure to light-dark were examined to determine the day-to-day reproducibility of the 24-h profile of cholesterol FSR and to test for the effects of prolonged

1 hospitalization and experimental proce- exposure t

z 1000

is

ii 900

Y I 0 800

!E w 700 z 2 B 600

5

x 500

ii

_-------- -------___ -A ,ss 55 A. 9-- . --- --. II----,-- ---WC- -8

--2-<-- --, -k--c;,- ---z--z- 8 =s=-c,“=-

--- C- v---- --. . - ---

-o----L---*9-‘- _-c-c Ir- -B . . .

‘-A--- ,---------------A

BL 1 2 3 4

DAYS

Fig. 3. Deuterium enrichment for the 5 simulated jet-lag subjects for the &day protocol. Dashed lines, individuals; solid lines, means + SD. Deuterium abundance before labeling averaged 141 + 1 ppm.


dures. Figure 4 shows the profiles for these two subjects on the baseline day and after 4 days in the CRC. These two subjects displayed two nadirs, 0700 and 1700 at baseline, but all within- and across-subject differences in the timings of the acrophase as well as nadirs were < 2 h. The overall waveshape of the diurnal profile appeared stable. Individual values for the 24-h mean FSR were 0.020 and O.O39/day on the baseline day and 0.014 and O.O32/day after 4 days in the CRC, respectively.

Profiles of Cholesterol FSR, Hormonal Levels, and Sleep on the Baseline Day

Figure 5 shows the mean profiles of cholesterol FSR, plasma cortisol, and plasma TSH for the baseline day. On the baseline day, the acrophase of cholesterol FSR occurred at 2325 t 0030 and the nadir at 0900 t 0040. The mean amplitude of the rhythm was 83 t 19%.

The profiles of plasma cortisol and TSH levels con- formed with classical descriptions (29) on the baseline day. The cortisol nadir occurred at 2325 t 0035 and the acrophase at 0925 t 0200. Levels of TSH increased in the late evening, reached an acrophase at 0005 t 0055, i.e., shortly after sleep onset, and then declined continuously. The mean amplitude of the rhythm was 27 t 8%.

Mean sleep efficiency on the baseline day was 89 t 7%. The sleep period was 424 t 16 min with 50 t 16 min awake, 243 t 13 min stages I + 11, 76 t 5 min stages III + IV, and 110 t 5 min REM sleep. During daytime recovery sleep on day 1, sleep efficiency was 87 t 10%

B t T T

BASELINE

-0.05 ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ 0700 1300 1900 0100 0700

TIME OF DAY

0.15 B L D

-I i\JI ? I

i DAY 31

-0.05 ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ 0700 1300 1900 0100 0700

TIME OF DAY

Fig. 4. Cholesterol fractional synthetic rate (FSR) during baseline day (top) and day 3 (bottom) in control subjects. Solid curves are the 121 moving averages. Stippled bar represents period of sleep.

h c I

8 g: 9

E 300

1

1900 0100 0700 1300 1900

TIME OF DAY

%l%i

1900 0100 0700 1300 1900

TIME OF DAY

Fig. 5. Baseline day values for cholesterol FSR (top), plasma cortisol (midde), and serum thyrotropin (TSH) (bottom) in the 5 simulated jet-lag subjects; means +_ SD. Stippled bar, period of sleep.

and the sleep period was 406 t 44 min, with 64 t 47 min awake, 240 t 46 min stages I + 11, 106 t 22 min stages III + IV, and 64 t 16 min stage REM.

Profiles of Cholesterol FSR, Hormonal Levels, and Sleep on Day 1

On day 1, which involved 12 h of sleep deprivation during the nighttime and recovery sleep during the daytime, the cholesterol FSR acrophase occurred at 2230 t 0030 and the nadir occurred at 1000 t 0100, i.e., essentially at the same clock times as on the baseline day (Fig. 6). The mean amplitude of the rhythm was not different from baseline, averaging 93 t 22%.

On day 1, the cortisol profile was not different from baseline, with a nadir at 2310 t 0025 and an acrophase at 0735 t 0030. The amplitude of the diurnal variation was also unchanged (76 t 9% on day 1 vs. 69 t 7% on the baseline day). In contrast, the TSH profile presented a marked increase in amplitude (from 27 t 4% on the


and that this change differed from that of the two controls (P < 0.001). Post hoc paired t-tests demonstrated that both the acrophase and the nadir of the profile of cholesterol FSR had changed significantly on shifted day 2 compared with baseline (P < 0.015 and P < 0.05, respectively), with the delay in acrophase averaging 5.0 t 0.7 h and the delay in the nadir averaging 5.4 t 1.9 h. The amplitude of the rhythm remained similar to baseline (85 t 14%). Contrasting with the significant shift in diurnal variation of cholesterol FSR, the profile of plasma cortisol remained syn- chronized to the baseline, rather than shifted, schedule. Indeed, the nadir occurred at 2400 t 0030 and the acrophase at 0750 t 0040, which was not significantly different from baseline. The amplitude tended to be lower than on the baseline day, averaging 54 t 8%, but the difference was not statistically significant. A trend for a partial adaptation of the acrophase of the TSH profile was apparent on day 2, as the acrophase occurred

h F

8 0.15

a 9 0.10

d 0.05

ki I- v) 3 0.00

B L D

1900 0100 0700 1300 19(

TIME OF DAY

0

cO. ? 6.0 . l-

DAY 1

B f% -0.05 L

V 1900 0100 0700 1300 1900

5oo :B L D

400 1 T T 1900 0100 0700 1300 1900

TIME OF DAY

Fig. 6. Sleep deprivation day (day 1) values for cholesterol FSR (top), plasma cortisol (middle), and serum TSH (bottom) in the 5 simulated jet-lag subjects; means 2 SD. Baseline day is indicated as a dashed line for comparison. Stippled bar, period of sleep.

baseline day to 51 t 4% on day 1, P < O.Ol), reflecting the well-known effects of nocturnal sleep deprivation on TSH secretion (1, 3, 20). The timing of the acrophase (0355 t 0100) was not quite significantly different from baseline (P = 0.1).

During daytime recovery sleep on day 1, sleep efficiency was 87 t 10% and the sleep period was 406 t 44 min, with 64 t 47 min awake, 240 t 46 min stages I + II, 106 t 22 min stages III + IV, and 64 t 16 min stage REM.

1900 0100 0700 1300 1900

TIME OF DAY

Profiles of Cholesterol FSR and Hormonal Levels on Day 2 oo3 .

1900 0100 0700 1300 1900

TIME OF DAY

Figure 7 illustrates the mean profiles of cholesterol FSR, plasma cortisol, and plasma TSH on the 2nd day of shifted sleep-wake cycle, light-dark cycle, and mealtimes. ANOVA indicated that the timing of both the acrophase and nadir of cholesterol FSR in the experimental groups changed with dav of the studv (P < 0.001)

Fig. 7. Day 2 of simulated jet-lag values for cholesterol FSR (top), plasma cortisol (middle), and serum TSH (bottom) in the 5 simulated jet-lag subjects; means f SD. Baseline day is indicated as a dashed line for comnarison. Stinnled bar. neriod of sleen.


A r B 0.15 .B L D ~~1.

the baseline schedule, but the second acrophase was ., ., .: :;: .: ,,., .: ..; : delayed 10.1 t 1.0 h from baseline (P < 0.01). The

a 2 0.10 :

amplitude of the TSH rhythm was significantly less T 0

z than under baseline conditions, averaging only 18 t 4%

6 . (P < 0.05). Th e d ecrease in amplitude appeared to be

a r

due to an increase in the nadir without change in the

8 acrophase.

. Sleep parameters on day 4 were similar to those

-005 5 . 1900 0100 0700 1300 1900

TIME OF DAY

‘** :B L D

ChoZesteroZ FSR and cortisol. On the baseline day there was a consistent negative cross correlation between the rhythms of cholesterol FSR and cortisol. The r value averaged -0.75 t 0.03 at a lag of +1.2 t 0.9 h (P < 0.05; cholesterol FSR leading cortisol). This strong inverse relationship persisted on shifted day 1, when the

1900 0100 0700 1300 1900 sleep-wake and light-dark cycles had been reversed, averaging - 0.79 t 0.06 at a lag of +l.O t 0.9 h

observed on the baseline day. Mean sleep efficiency was 82 t 7%. The sleep period was 389 t 34 min with 79 t 30 min awake, 215 t 22 min stages I + II, 70 t 12 min stages III + W, and 111 t 1 min REM stage.

Cross-Correlation Analysis

. /- -A

1.0 : DAY 4

0.0 ” ” ’ ” ” ” ” ” ” ” ” ” ” 1900 0100 0700 1300 1900

TIME OF DAY

Fig. 8. Day 4 of simulated jet-lag values for cholesterol FSR (top), plasma cortisol (middle), and serum TSH (bottom) in the 5 simulated jet-lag subjects; means 2 SD. Baseline day is indicated as a dashed line for comparison. Stippled bar, period of sleep.

at 0630 t 0130, i.e., 6.3 t 2.1 h later than on the baseline schedule (NS, P = 0.1). The amplitude was similar to that observed on the baseline day, averaging 29?2%.

Profiles of Cholesterol FSR, Hormonal Levels, and Sleep on Day 4

Figure 8 shows the mean profiles of cholesterol FSR, cortisol, and TSH on shifted day 4. The acrophase and nadir of the FSR profiles were significantly shifted from baseline (P < 0.015 and P < 0.015, respectively), with

(P < kOvs>. On day 2 and day $ cross correlations between the rhythms of cholesterol FSR and cortisol were no longer significant (day 2 -0.23 t 0.06 at a lag of +4.2 t 1.2 h;day4 -0.23 t 0.05at alagof +0.8 t 1.4 h), reflecting the dissociation of these two rhythms during adaptation to the 12-h simulated time zone shift as well as differences in individual rates of adaptation.

ChoZesteroZ FSR and TSH. Under baseline conditions, temporal changes in cholesterol synthesis were less consistently correlated with the profile of plasma TSH than with that of cortisol. The largest coefficient of cross correlation between the cholesterol FSR and TSH rhythms on the baseline day was +0.53 t 0.09 at a lag of - 3.8 t 1.4 h (TSH leading cholesterol FSR), which was not statistically significant. However, on shifted day 1, i.e., during sleep deprivation and recovery sleep, a significant positive correlation between the two rhythms was detected, with a maximal coefficient of cross correlation of +0.71 t 0.06 at a time lag of -5.2 t 0.06 h (P < 0.05). On day 2 and day 4, the cross correlations between the two rhythms were inconsistent and no longer significant, indicating a dissociation of the two rhythms. On day 2, the maximum coefficient of cross correlation was +0.33 t 0.02 at a lag of + 1.3 t 0.8 h in 4 subjects and -0.28 at a lag of +6.0 in the fifth subject. On day 4, the maximum coefficient was +0.30 t 0.04 at a lag of -0.3 t 0.3 h in four subjects and zero lag in the fifth subject.

- 0.30 with

delay averaging 9.6 ? 1.7 h for the acrophase and 14.8 t 0.5 h for the nadir. Partial adaptation of the cortisol

DISCUSSION

profile to the simulated time zone shift was evident, as The present results provide an unequivoca the nadir was delayed by 5.4 t 1.5 (P < 0.06) and the tion of the existence of a marked diurnal rh

demonstra- tihmicity in

acrophase was delayed 5.6 t 2.1 h from baseline (NS). human de novo cholesterol synthesis that displays little Rhythm amplitude was dampened compared with base- between-individual variability under controlled condi- line conditions (averaging 49 t 6 vs. 69 t- 7%, NS). The tions. Under baseline conditions, the intrasubject vari- TSH profile exhibited two, rather than one, acrophases, abilities in the timing of the zenith and nadir were occurring at 2340 t 0015 and 1020 t 0030, respectively. remarkably stable, ranging from 2200 to 0100 and 0700 The first acrophase appeared to be a reestablishment of to 1130, respectively. The amplitude was quite large but


somewhat more variable (range 28-142%), with the mean diurnal variation in vivo cholesterol FSR being O.OO/day in the morning nadir and O.OS/day at the nighttime acrophase. These minimal and maximal rates of cholesterol synthesis are consistent with previous estimations obtained under less controlled conditions and with a lower sampling frequency (17). Furthermore, the amplitude of the diurnal variation in cholesterol FSR by direct measurement in the present study is similar in magnitude to that reported for plasma levels of mevalonate (15,22).

In the current study, the mean maximal rate of cholesterol synthesis under baseline conditions occurred at midnight-and the minimal rate occurred in the mid- morning. Our estimation of the timing of the peak of the diurnal variation is thus considerably earlier than the 0600 zenith reported in the first investigation of the diurnal rhythm of human cholesterol synthesis using deuterium incorporation (17). However, our estimation is nearly identical to that reported in a later study using the same technique (15). Moreover, the present findings are compatible with two previous studies of the diurnal variations of plasma cholesterol precursor levels, which both reported maximal values between midnight and 0400 (18, 22). These latter small discrepancies may reflect differences in sampling frequency. In the present investigation, the 2-h sampling frequency was more frequent than in previous studies to obtain the highest peak resolution with the least amount of measurement noise (unpublished data). However, differences in study conditions related to activity level, diet composition, and mealtimes could also account for discrepancies in the timing of the zenith and nadir. The current protocol was specifically designed to maximize interindividual synchronization with respect to these variables before our controlled intervention.

A striking negative correlation was found between the rhythm of cholesterol synthesis and that of plasma cortisol during both the baseline day and the first day of simulated jet lag, suggesting that the control of cholesterol synthesis by circadian timing could be mediated by cortisol. In this respect, our findings in the human are quite similar to those reported in laboratory animals. Adrenalectomy in rats has been demonstrated to decrease (19) or abolish (7) the rise in enzyme activity that occurs during the dark phase. A single injection of hydrocortisone administered to adrenalectomized rats 3 h before the expected zenith in enzyme activity resulted in a twofold increase in the activity of the enzyme at the zenith, to values similar to that observed in controls (19). However, administration of hydrocortisone 3 h before the expected minimum did not result in a change in activity of the enzyme at the nadir (19), suggesting that glucocorticoids are not the only factor triggering induction of the enzyme. These animal findings, support- ing a positive association between corticosteroids and cholesterol synthesis, contrast with the observation in the present study of a negative correlation. However, in human skin fibroblasts, physiological concentrations of hydrocortisone were reported to suppress, rather than activate, the cholesterol synthetic pathway (12).

The first day of the simulated time shift included nighttime sleep deprivation and daytime recovery sleep, and a reversal of the light-dark cycle. Under conditions of shifted. sleep-wake and light-dark cycles without concomitant shift of mealtimes (shifted day I), the diurnal pattern of cholesterol synthesis was essentially unaltered, demonstrating that the rhythm is not acutely regulated by the sleep-wake cycle or lighting conditions. In addition, results indicate that hormones that are strongly dependent on sleep (27, ZS), such as growth hormone (GH), are unlikely to have immediate effects on cholesterol synthesis. The observation that the abrupt displacement of sleep, and therefore of sleep-related GH release, is not associated with detectable changes in cholesterol synthesis is in agreement with the study by Boyle et al. (2), who measured plasma mevalonate and GH levels in GH-deficient and -sufficient patients and found no correlation between peak nocturnal mevalonate concentrations, fasting mevalonate concentrations, and GH levels. It is possible, however, that the effects of sleep-wake reversal on cholesterol FSR rhythm might take longer than 1 day to appear. Over the 4 shifted days, however, comparison of the rates of adaptation of the rhythm of cholesterol synthesis and of the rhythm of cortisol, which is primarily driven by the central circadian signal and only slightly influenced by mealtimes (29), provided evidence that neither sleep-wake cycle nor the circadian clock was the primary factor in synchroniz- ing the rhythm of cholesterol synthesis. Conclusions based on these comparisons were further strengthened by examining the rate of adaptation of the TSH rhythm, which is influenced by both circadian rhythmicity and sleep, but not by meal intake.

The dissociation between the rhythms of cholesterol synthesis from these of cortisol and TSH in the course of adaptation to jet lag does not conclusively exclude a role for the circadian clock in the control of cholesterol synthesis. To do so requires a demonstration that the rhythm of cholesterol synthesis does not persist when caloric intake is continuous, rather than distributed in three meals. In this regard, Jones et al. (14) have demonstrated a diminution of the rhythm when enteral intake was divided over six meals taken at 4-h intervals. This study by Jones et al., however, only involved a small number of subjects, and a small rhythm may have escaped statistical significance. Further studies during continuous enteral nutrition or constant glucose infu- sions will be necessary to address this issue.

On the baseline day, the daily maximum of cholesterol synthesis was coincident with peak levels of TSH, a hormone that is normally inhibited by nocturnal sleep. As anticipated, a more than twofold increase in the amplitude of the TSH rhythm was observed during sleep deprivation (1,3,21). This major alteration in the profile of TSH concentrations was associated with a modest elevation of the acrophase of the rhythm of cholesterol synthesis, and these concomitant changes in both rhythms were reflected in an increase in their coefficient of cross correlation. These findings raise the possibility that TSH may exert an effect on cholesterol synthesis, particularly during sleep deprivation. During normal


sleep-wake conditions, the diurnal variation in thyroid hormone secretion is of low amplitude and may even be undetectable (1,29). However, during sleep deprivation, a well-defined nocturnal increase in thyroid hormone levels parallels the elevation of TSH levels (29). Because the activity of HMG-CoA reductase has been demonstrated to be affected by thyroid hormone (10, ZO), it is conceivable that the activation of the pituitary-thyroid axis associated with sleep deprivation on the 1st day of the simulated time zone shift exerted a modest modula- tory influence on cholesterol synthesis.

In contrast to the absence of a shift in the diurnal rhythm of de novo cholesterol synthesis on day 1 when sleep and the light-dark cycles were altered, the timing of the nadir and zenith of cholesterol synthesis was altered on the 1st day after the meal schedule was shifted by 12 h (day 2). This is similar to studies performed in rats (8). Furthermore, because the diurnal rhythm in humans is suppressed by feeding six meals at 4-h intervals around the clock (14) and cholesterol FSR is suppressed on the 1st day of fasting (16), it is likely that mealtime plays a major role in the regulation of the diurnal variation of de novo cholesterol synthesis. Fur- ther studies in which only mealtime is varied, however, should be performed.

In conclusion, the present study demonstrates that de novo cholesterol synthesis varies dramatically across the day, indicating that cholesterol synthesis in humans is a tightly regulated system. This 24-h rhythm is not acutely dependent on the sleep-wake cycle. Mealtime appears to play a major role in determining the timings of the daily maximum and minimum synthetic rate. Further studies are needed to determine the importance of the mealtime for cholesterol homeostasis in response to dietary cholesterol.

We thank Dr. Neil Scherberg and the staff of the Thyroid Function Laboratory for TSH determinations, William Pugh for cortisol assays, John Lukens for measurements of cholesterol levels, and Rachel Leproult for help with computerized data analysis. We gratefully acknowledge the skillful assistance of Jacqueline Imperial, Beena Loharikar, and the staff of the University of Chicago Clinical Research Center.

This work was partially supported by National Institutes of Health Grants HL-45574, DK-26678, DK-41814, and RR-00055 and Grant F49620-94-1-0203 from the Air Force Office of Scientific Research.

Address for reprint requests: D. A. Schoeller, Dept. of Medicine, MC 4080, Univ. of Chicago, 5841 South Maryland Ave., Chicago, IL 60637.

Received 30 November 1994; accepted in final form 6 April 1995.

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jet lag and cholesterol synthesis

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