Volume 25, Issue 22, Pages 3004-3010 (November 2015) Morning Circadian Misalignment during Short Sleep Duration Impacts Insulin Sensitivity  Robert H. Eckel, Christopher M. Depner, Leigh Perreault, Rachel R. Markwald, Mark R. Smith, Andrew W. McHill, Janine Higgins, Edward L. Melanson, Kenneth P. Wright  Current Biology  Volume 25, Issue 22, Pages 3004-3010 (November 2015) DOI: 10.1016/j.cub.2015.10.011 Copyright © 2015 Elsevier Ltd Terms and Conditions

Figure 1 Experimental Protocol Example protocol showing one of the condition orders tested starting with 3 baseline days with 9-hr sleep opportunities followed by 5 days in the 5-hr condition and then 5 days in the 9-hr condition. Equal numbers of subjects completed 5- and 9-hr conditions first, in randomized order. Data represent recordings from the Actiwatch-L recorder, with activity denoted by black ticks and light exposure denoted in yellow. Data are double plotted with successive days plotted both next to and beneath each other; clock hour is indicated on the abscissa. Sleep timing was based on individual habitual sleep schedules. During the 5-hr insufficient sleep condition, sleep was restricted by delaying bedtime and advancing wake time by 2 hr each. Oral glucose tolerance tests were administered on study days 2 (baseline), 7, and 12 (green boxes). Intravenous glucose tolerance tests were administered on study days 4 (baseline), 9, and 14 (red boxes). Hourly blood samples were collected across 24 hr on study days 3 (baseline), 8, and 13 for assessment of insulin, glucose, and melatonin (24-hr circadian phase assessments). Current Biology 2015 25, 3004-3010DOI: (10.1016/j.cub.2015.10.011) Copyright © 2015 Elsevier Ltd Terms and Conditions

Figure 2 Insulin Sensitivity at Baseline and during Adequate and Short Sleep Opportunities OGTT Matsuda ISI and IVGTT SI by condition (A and C, n = 16) and condition order (B and D, n = 8 each condition order), respectively. OGTT Matsuda ISI, Oral Glucose Tolerance Test Matsuda Insulin Sensitivity Index [1 / (mmol/l)2 × (pmol/l)2]. IVGTT SI, Intravenous Glucose Tolerance Test Insulin Sensitivity [(mU/l)–1 × min–1]. ∗p < 0.033 (planned comparison, one-tailed dependent t test left panel), ∗p < 0.05 (planned comparison, two-tailed dependent t test right panel). Horizontal lines indicate comparison between conditions at the ends of line. Order of bars in (B) and (D) show order of conditions. Data are mean ± SEM. See also Figures S1–S3. Current Biology 2015 25, 3004-3010DOI: (10.1016/j.cub.2015.10.011) Copyright © 2015 Elsevier Ltd Terms and Conditions

Figure 3 Hourly Plasma Melatonin Curves before and after Scheduled Wake Time Average hourly melatonin data are shown for each condition with hours from scheduled wake time (0 and dashed vertical line) on the abscissa; negative values denote hours before and positive values denote hours after scheduled wake time (n = 16). Dashed horizontal lines indicate the approximate timing of the OGTT and IVGTT tests, which occurred on the days before and after melatonin assessment, respectively. pg/ml, picograms per milliliter. OGTT, oral glucose tolerance test. IVGTT, intravenous glucose tolerance test. Data are mean ± SEM. Current Biology 2015 25, 3004-3010DOI: (10.1016/j.cub.2015.10.011) Copyright © 2015 Elsevier Ltd Terms and Conditions

Figure 4 Associations between Insulin Sensitivity and Insulin Secretion with Circadian Timing Association between OGTT Matsuda ISI (A), IVGTT SI (B), OGTT insulin AUC (C), and IVGTT AIRg (D) with circadian timing of DLMOff25% relative to wake time across conditions. The vertical dashed line represents scheduled wake time. A negative value of DLMOff25% relative to wake time indicates a melatonin offset prior to scheduled wake time and a positive value of DLMOff25% relative to wake time indicates a melatonin offset after scheduled wake time. Symbols represent individual subjects. β represents the beta estimate of the association between dim-light melatonin offset relative to scheduled wake time and OGTT Matsuda ISI (A), IVGTT SI (B), OGTT insulin AUC (C), and IVGTT AIRg (D) from the linear mixed model. OGTT Matsuda ISI, Oral Glucose Tolerance Test Matsuda Insulin Sensitivity Index [1 / (mmol/l)2 × (pmol/l)2]. IVGTT SI, Intravenous Glucose Tolerance Test Insulin Sensitivity [(mU/l)–1 × min–1]. AUC, area under the curve (mg/dl × min). AIRg, acute insulin response to glucose (mU × l–1 × min). DLMOff25%, dim-light melatonin offset. p values for the associations can be found in the main text results. Current Biology 2015 25, 3004-3010DOI: (10.1016/j.cub.2015.10.011) Copyright © 2015 Elsevier Ltd Terms and Conditions