<p>(A) Glucose tolerance test. Glucose concentration (mg/dL) in whole blood of Ate1-containing mice (n = 15; black curve) and Ate1-deficient mice (n = 11; red curve), at different times after a bolus of glucose by gavage, following a 16-hr fast. Glucose was administered at time zero. Error bars indicate ±SD. (B) Fasting blood glucose levels. Average blood glucose levels (mg/dL) in Ate1-containing mice (n = 15; black bar) and Ate1-deficient mice (n = 11; red bar), with measurements shortly before glucose gavage (after a 16-hr fast) and 6 hr after the gavage in A. Standard deviations are indicated. Statistical analysis was performed using an unpaired t-test (p<0.04). (C) Average daily energy consumption (kcal/gm of body weight) for Ate1-containing mice (n = 5; black curve) and Ate1-deficient mice (n = 3; red curve), with measurements from 1 week prior to tamoxifen (TM) treatment. Vertical arrow indicates the beginning of a 5-day TM treatment. Error bars indicate ±SD. (D) Relative efficiencies of the import of <sup>14</sup>C-amino acids and <sup>14</sup>C-peptides from gastrointestinal tract in Ate1-containing mice (black bars) versus Ate1-deficient mice (red bars). Shown here are representative comparisons of the retention of <sup>14</sup>C (in cpm/gm) in the brains, livers, spleens, kidneys, and hearts of indicated mice 16 days after gavage with a single bolus of <sup>14</sup>C-labeled proteins (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007757#s4" target="_blank">Materials and Methods</a>). Mice were gavaged 26 days after TM treatment. (E) Comparison of resting metabolic rate (RMR) (measured in O<sub>2</sub> (ml) consumed per kg of body weight per min) for Ate1-containing mice (n = 6; black bar) versus Ate1-deficient mice (n = 6; red bar). Standard deviations are indicated in E and F. Statistical analysis was performed using an unpaired t-test (p<0.008). (F) Comparison of the respiratory exchange ratio (RER), measured as CO<sub>2</sub> (in ml) per ml of O<sub>2</sub>, for Ate1-containing mice (n = 6; black bar) and Ate1-deficient mice (n = 6; red bar) mice. No statistically significant difference in RER was observed. (G) RT-PCR analyses of <i>AgRP, MCH, HPY,</i> and <i>POMC</i> mRNA levels in the hypothalami of TM-treated Ate1-containing mice (Sets 1 and 4) versus Ate1-deficient mice (Sets 2 and 3). Set 1, <i>Ate1<sup>flox/flox</sup></i> (+/+); Set 2, <i>Ate1<sup>flox/flox</sup></i>;<i>CaggCreER</i> (−/−); Set 3, <i>Ate1<sup>flox/flox</sup></i>;<i>CaggCreER</i> (−/−); Set 4, <i>Ate1<sup>flox/+</sup></i>;<i>CaggCreER</i> (+/−). In sets 1 and 2, hypothalami were isolated 93 days after TM treatment. In sets 3 and 4 hypothalami were isolated ∼1 year after TM treatment. Sloping triangles indicate decreasing inputs (by 2-fold) of total RNA.</p
