gender: male subjectid: RAS14 age (yrs): 21 height (m): 1.82 weight (kg): 82.3 bmi (kg/m2): 24.8 time of sampling: 7 AM (7h) experimental condition: circadian misaligned fasting glucose (mmol/l): 5.164 fasting insulin (uu/ml): 12.6 fasting ffa (umol/l): 354.54 fasting triglycerides (mmol/l): 1.13
Treatment protocol
Pre-study conditions: Seven days prior to the study periods, participants were instructed to maintain a standardized normal lifestyle, including (trying to) sleep from 11 PM until 7 AM and consuming meals at regular times, consistent with the times during the study (breakfast, lunch, snack and dinner at 8 AM, 12:30 PM, 3 PM and 8 PM, respectively). In addition, participants were asked to abstain from consumption of caffeine and alcohol during those 7 days. Three days before the study periods, participants were instructed not to perform physical exercise. Two days before each in-laboratory study protocol, participants were provided with meals to ensure standardized distribution of calories and macronutrients (see below). Compliance to the prescribed lifestyle was monitored with food- and sleep diaries and by a light-detecting wrist accelerometer (Actiwatch Spectrum, Philips-Respironics, Murrysville PA, USA). Study conditions: In the study, each participant underwent one control protocol and one circadian misalignment protocol, in a randomized, crossover fashion, with a wash-out of 3 to 9 weeks. Prior to the first study period, the participants were not aware of the order of the study periods. During their stay, the participants resided in a private respiration chamber; a small room with a bed, toilet, sink, desk, chair, TV and computer. In the respiration chamber environmental conditions were tightly controlled, including dim-light (4 lux in the horizontal angle of gaze) during wake phases and darkness during sleep opportunities, no devices that can display time (e.g. watch, mobile phone, laptop, tablet), no internet and no live television or radio. The screen emittance of the TV and computer in the respiration chamber were adapted to not exceed the permitted light intensity of 4 lux and could be used to work, listen to music or watch movies. During their stay, participants were isolated from external time cues and could only be in contact with the researchers. For specific study procedures (placement of intravenous canula, indirect calorimetry, skeletal muscle biopsy and the hyperinsulinemic euglycemic clamp), participants were transported to an adjacent clinical room in a wheelchair, during which they wore a blindfold and earplugs to prevent excess light and possible audiovisual signals that might indicate whether it was day or night. The clinical room had similar dim-light conditions. During procedures for which light was required (placement of intravenous canula, muscle biopsy), participants wore welding goggles shade 5 (Uvex Ultravision 9301-245, Uvex, Fürth, Germany) to limit light exposure. During each study period, two skeletal muscle biopsies were obtained according to the Bergström method (PMID: 5584523) from the m. vastus lateralis under local anasthesia (1% lidocaine, without epinephrine) at 7 AM and 7 PM. Biopsies were taken moving from distal to proximal, alternating between the left or right leg. The leg of the first biopsy was randomized and subsequent biopsies were taken from the other leg. Muscle fiber type was similar between control and misalignment condition. Samples were snap-frozen in liquid nitrogen, and stored at -80 degrees centigrade until RNA isolation.
Growth protocol
Fourteen healthy lean young men (Age: 22.4 ± 2.8 years; BMI: 22.3 ± 2.1 kg/m2; Mean ± SD) participated in the study. Participants were non-smokers, had no active diseases, used no medication and did not engage in exercise for more than 3 hours per week, verified by questionnaires. In addition, participants reported regular bedtimes (11 PM ± 2 hours), regular sleep duration of 7-9 hours, did not perform shift-work or traveled across more than one time zone in the last three months and were not definite morning larks or night owls, assessed with a morningness-eveningness questionnaire (MEQ-SA: 54 ± 7, Mean ± SD). The study was conducted in accordance with the principles of the declaration of Helsinki, approved by the Ethics Committee of the Maastricht University Medical Center and monitored by the Clinical Trial Center Maastricht. All participants provided written informed consent. The included measurements were performed between February 2016 and February 2017. The study was registered at clinicaltrials.gov with identifier NCT02580513.
Extracted molecule
total RNA
Extraction protocol
Total RNA was prepared from the muscle biopsies (50 mg) using TRIzol reagent, whereafter purified total RNA was isolated using Qiagen RNEasy columns. RNA integrity was checked on chip analysis (Agilent 2100 bioanalyzer, Agilent Technologies, Amsterdam, the Netherlands) according to the manufacturer's instructions. RNA was judged as suitable for array hybridization only if samples exhibited intact bands corresponding to the 18S and 28S ribosomal RNA subunits, and displayed no chromosomal peaks or RNA degradation products (RNA Integrity Number > 8.0).
Label
biotin
Label protocol
Purified total RNA (100ng per sample) was labeled with the Whole-Transcript Sense Target Assay (Affymetrix, Santa Clara, CA, USA; P/N 900652).
Hybridization protocol
Hybridization and washing of the Affymetrix GeneChip Human Gene 2.1 ST peg arrays were performed on a GeneTitan Instrument (Affymetrix, Santa Clara, CA) according to the manufacturer's recommendations.
Scan protocol
Arrays were scanned on an Affymetrix GeneTitan instrument (Affymetrix, Santa Clara, CA).
Data processing
Expression estimates were calculated applying the RMA algorithm in the Bioconductor library 'Oligo' (v1.42.0).
