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

Study design schematic.

<p>Twenty percent weight reduction was achieved by feeding mice 1g of chow daily. During the weight maintenance phase, food intake was increased to 2-3g per day per mouse (the amount of food was adjusted daily when % of initial body weight of a mouse deviated from 80% by more than 2%). Calorically-restricted mice were provided with food twice daily, 1/3 of the total daily calories in the morning (09:00–9:30h) and 2/3 in the evening (18:00–18:30h). Body weight and food intake were monitored daily. During the second week of weight maintenance, mice were placed individually in metabolic cages to assess their energy expenditure (EE; TSE calorimetry system). They were then released to <i>ad libitum</i> feeding and EE was measured for another week. Mice were monitored for eight weeks until the body weight of the previously calorie restricted group reached that of with the never-restricted controls at which point mice were sacrificed.</p

Correlations of energy expenditure with body composition in AL and CR mice.

<p>Regression of (A, C) lean mass and (B, D) fat mass against (A, B) total and (C, D) resting energy expenditure in the AL and CR groups of mice during the weight maintenance segment of the CR phase.</p

Body temperature of AL and CR mice.

<p>Body temperature of mice fed <i>ad libitum</i> chow throughout the study (AL) and mice calorically restricted to 80% of initial body weight (CR) measured during the weight maintenance segment of the CR phase.</p

Correctly targeted ESC clones are pluripotent.

<p>Immunofluorescence of correctly targeted ESC clone rs1421085 (C/C) showing pluripotency molecular markers homeobox transcription factor NANOG, octamer-binding homeodomain transcription factor 4 (OCT4), glycoprotein TRA-1-80, Stage-Specific Embryonic Antigen-4 (SSEA-4), and transcription factor SRY (sex determining region Y)-box 2 (SOX2). Pluripotency markers are shown in green, nuclei are counterstained with DAPI.</p

Quality control measures of correctly targeted clones.

<p>(A, B) Results from Sanger sequencing. The SNPs rs9940128 (near rs1421085) and rs4783819 (near rs8050136) were amplified and sequenced in the same read to control for possible long deletions in the correctly targeted ESC clones. (C) Two representative karyotypic images. All correctly targeted clones tested displayed a normal karyotype.</p

Schematic representation of the <i>FTO</i> genomic locus (chr16:53,703,963–54,121,941).

<p>(A) SNPs rs1421085 (C/T) and rs8050136 (A/C) are located in the first intron of <i>FTO</i>. (B, C) CRISPR/Cas9 technology was employed to convert ESC line H9 (heterozygous for both SNPs) to homozygosity for both alleles at rs1421085 (C/C or T/T) or rs8050136 (C/C or A/A). Positions of gRNA, PAM sequence and ssODN are indicated by thick lines in blue, purple and black, respectively. SNPs are given in green. Predicted Cas9 cut sites are indicated by red arrow heads.</p