ICE#1, effect of 1 hour and 4 hours of cold exposure three times per week.

<p>Tissue weights, mRNA levels, and serum analytes were taken at the end of the experiment. TEE was calculated as described in Materials and Methods using the energy balance method over the full 77 days of the experiment. Gene expression values are reported relative to CON. Data are mean ±SE, N = 8/group. Levels not connected by same letter are significantly different (P<0.05).</p><p>includes adherent WAT.</p

Effects of AM251 and cold exposure.

<p>Mice were administered vehicle or AM251 (3 mg/kg/day) by oral gavage. A: Caloric intake and B: body weight were measured three times per week on days of cold exposure. C: Mice at 30°C were administered CL316243 (100 µg/kg) at time 0 as described in Experimental Procedures (the prior AM251 was given at −24 hours). Inset shows the delta TEE, calculated as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085876#pone-0085876-g002" target="_blank">Figure 2</a>. 2-way ANOVA showed significant temperature (p<0.01) and drug (p<0.05) effects with no significant interaction (p = 0.62). D: Intra-peritoneal glucose tolerance tests (1 g/kg) were performed in overnight-fasted mice of the indicated treatment group two days after cold exposure. AUC numbers are in mg/dl•minute. E. Insulin tolerance test. Insulin (0.75 U/kg) was injected and blood glucose measured at the indicated times. GTT =  intraperitoneal glucose tolerance test. CL =  CL316243 experiment. ITT =  insulin tolerance test. AM251 was administered 24 h prior to the GTT and ITT. In D & E, a poor-responding outlier in the CON AM group was removed from the analysis. If included in the ipGTT AUC, this group's AUC is 29304 ±3382 mg/dl min and the CON AM significantly different from the ICE AM group. All data are mean ±SE. N = 6/group. Levels not connected by same letter are significantly different (P<0.05).</p

ICE#2, effect of 4 hours and 8 hours of cold exposure three times per week.

<p>Tissue weights, mRNA levels, and serum analytes were taken at the end of the experiment. TEE was calculated as described in Materials and Methods using the energy balance method over the full 74 days of the experiment. Gene expression values are reported relative to CON. Data are mean ±SE, N = 8/group. Levels not connected by same letter are significantly different (P<0.05).</p

Tissue 2-deoxyglucose uptake.

<p>Mice in the ad lib fed state were administered [¹⁴C]2-deoxyglucose 1 hour prior to euthanasia. Specifically, this was at 22°C in the CON group, at the onset of 4°C in the 1 hour group, and 3 h into the 4°C treatment in the 4 hour group. Data are mean ±SE, N = 8/group. Levels not connected by same letter are significantly different (P<0.05).</p

Transient improvement in glucose tolerance by cold exposure.

<p>In ICE#1 (top) and ICE#2 (bottom), intra-peritoneal glucose tolerance tests (1 g/kg) were performed in the overnight-fasted mice. In ICE#1, the ipGTT was conducted the day after cold exposure, while in ICE#2 it was conducted on the second day following cold exposure. The inset shows the mean area AUC in mg/dl•min ±SE, N = 8/group. Levels not connected by same letter are significantly different (P<0.05).</p

Cold exposure increases food intake, but not body weight.

<p>A: Caloric intake and B: body weight during ICE#1 (top) and ICE#2 (bottom) experiments were measured three times per week on days of cold exposure. Timing of the intra-peritoneal glucose tolerance tests (GTT) and CL316243 treatment (CL) are indicated. Data are mean ±SE, N = 8/group. The 8 h group had higher food intake as assessed by repeated measures ANOVA (p<0.05).</p

Food intake, plasma glucose and insulin in CR and AL mice.

<p>(A) Mean 24h food intake ±SEM (g) and (B) Cumulative food intake over 8 weeks of body weight re-gain in mice fed <i>ad libitum</i> chow throughout the study (AL) and mice calorically restricted to 80% of initial body weight then released to <i>ad libitum</i> feeding. (C) Mean glucose and (D) insulin ±SEM in <i>ad libitum</i> fed (AL) or calorically restricted (CR) mice measured at 12 weeks of age while CR mice were calorically restricted to maintain 80% of initial body weight. (E) Regression of circulating insulin concentrations against fat mass in the AL and CR groups of mice at 11 weeks of age while CR were weight stable at the reduced body weight. P values: ***<0.001.</p

Body weight and food intake of AL and CR mice in a pilot study.

<p>(A) Mean body weight ±SEM (g) and (B) Mean 24h food intake in mice fed <i>ad libitum</i> throughout the study (AL) and mice calorically restricted to 80% of initial body weight then released to <i>ad libitum</i> feeding. P values: *<0.05, **<0.01.</p

Total energy expenditure and respiratory exchange ratio.

<p>(A) Total energy expenditure after release from calorie restriction in mice fed <i>ad libitum</i> chow throughout the study (AL) and calorically restricted (CR) mice. TEE post-restriction was calculated using the energy balance equation: TEE = FI − (Δ somatic Fat Energy + Δ somatic Fat−Free Energy). (B) average respiratory exchange ratio (RER) measured at each time interval and (C) average 24-hour RER during the day and (D) and at night during and post calorie restriction in mice fed <i>ad libitum</i> chow throughout the study (AL) and mice calorically restricted then released to <i>ad libitum</i> feeding. P values: **<0.01, ***<0.001.</p

Energy expenditure and activity of AL and CR mice.

<p>(A) Energy expenditure during calorie restriction in mice fed <i>ad libitum</i> chow throughout the study (AL) and mice calorically restricted to 80% of initial body weight (CR). Energy expenditure during calorie restriction was measured in the TSE metabolic chambers. Included are the following: TEE–total energy expenditure, REE–resting energy expenditure, NREE–non resting energy expenditure and torpor suppression. (B) Physical activity in AL and CR mice during CR and after release to <i>ad libitum</i> feeding. Activity was measured in the TSE system. Regression of instantaneous TEE as a function of movement (C) during the day and (D) at night in mice fed <i>ad libitum</i> chow throughout the study (AL), mice calorically restricted to 80% of initial body weight (CR) and the CR group after release to <i>ad libitum</i> feeding. P values: *<0.05, ***<0.001.</p