TRAMP^fmsmic-1 mice survive longer and have smaller prostate tumors than TRAMP mice.

<p>Overall survival of individual mice from birth to death is shown (panel A). The survival data for TRAMP (—▴—) and TRAMP^fmsmic-1 (—Δ—) mice were plotted using the Kaplan-Meier method. The log-rank statistic for median survival time is shown. The genitourinary (GU), seminal vesicle (SV) and prostate tumor sizes in TRAMP^fmsmic-1 and TRAMP mice (n = 15/group) sacrificed at 8, 17, 25 and 33 weeks of age are shown in panel B, C and D. The GU (panel B), SV (panel C) and prostate weights (panel D) are presented as mean mg weight/g body weight ± SEM. p values are shown as *p<i><</i>0.05; **p<i><</i>0.01; ***p<i><</i>0.001.</p

MIC-1/GDF15 increases metastasis.

<p>(A) Comparison between proportion of TRAMP (n = 30) and TRAMP^fmsmic-1 (n = 30) mice having distant organ metastasis. The data is analyzed by Chi-square test. (B) Comparison between proportion of TRAMP (n = 59) and TRAMP^fmsmic-1 (n = 33) mice with metastasis at 18–40 weeks, a period where TRAMP and TRAMP^fmsmic-1 mice reach ethical end points at the same rate. The data is analyzed by Chi-square test. (C) Comparison between number of lung tumor colonies in MIC-1^fms mice and control C57BL/6 mice injected intravenously with TC1-T5 cells. The data is analyzed by Unpaired <i>t</i> test. The p values for Chi-square test and Unpaired <i>t</i> test are shown as ***p<i><</i>0.001.</p

MIC-1/GDF15 overexpression improves glucose tolerance and response to insulin.

<p>A. Blood glucose concentrations in response to i.p. glucose injection (1 g/kg) in normal chow-fed mice overexpressing MIC-1/GDF15 (MIC-1^fms) and control mice (MIC-1^+/+) at 23 weeks of age. B. Area under the curve calculated from the glucose tolerance test in (A). C. Blood glucose concentrations in response to i.p. insulin injection (1 U/kg) in normal chow-fed MIC-1^fms and MIC-1^+/+ mice at 24 weeks of age. Data are means ± SEM of 5 female mice per group. *p&lt;0.05, **p&lt;0.01 and ***p&lt;0.001 for the difference between genotypes.</p

TRAMP^fmsmic-1 prostate tumors have lower histological grades.

<p>Prostates of 15 TRAMP and 15 TRAMP^fmsmic-1 mice were excised at week 8 (panel A), 17 (panel B), 25 (panel C), and 33 (panel D). Serial sections were graded and scores for each grade were averaged for all the mice in the group. The graph represents the mean proportion of the prostate from all the mice in the group having each different pathologic grade, scored between 1 and 6± SEM. Numbers on the bars show number of mice in the group having a particular grade. p values are shown as *p<i><</i>0.05; **p<i><</i>0.01; ***p<i><</i>0.001.</p

MIC-1/GDF15 overexpression improves glucose tolerance in mice on a high fat diet.

<p>A. Blood glucose concentrations in response to i.p. glucose injection (1 g/kg) in mice overexpressing MIC-1/GDF15 (MIC-1^fms) and control mice (MIC-1^+/+) at 23 weeks of age, after 13 weeks on a high fat diet. B. Area under the curve calculated from the glucose tolerance test in (A). C. Blood glucose concentrations in response to i.p. insulin injection (1 U/kg) in MIC-1^fms and MIC-1^+/+ mice at 24 weeks of age, after 14 weeks on a high fat diet. Data are means ± SEM of 5 female mice per group. *p&lt;0.05, **p&lt;0.01 and ***p&lt;0.001 for the difference between genotypes.</p

MIC-1/GDF15 overexpression reduces body weight, adiposity and food intake without altering metabolism.

<p>A. Body weight of mice overexpressing MIC-1/GDF15 (MIC-1^fms) and control mice (MIC-1^+/+) from 11 to 24 weeks of age, represented as 0–13 weeks on the normal chow diet. B–E. Absolute and relative (as a percent of body weight) fat and lean mass as determined by dual energy X-ray absorptiometry (DXA) in normal chow-fed MIC-1^fms and MIC-1^+/+ control mice at 26 weeks of age. F–I Mass of white adipose tissue (WAT) and interscaptular brown adipose tissue depots as absolute weight (F, H) or normalized to body weight (G, I) in normal chow-fed MIC-1^fms and MIC-1^+/+ control mice at 26 weeks of age. i, inguinal; p, periovarian; r, retroperitoneal and m, mesenteric WAT depots. J–K. Spontaneous (J) and cumulative 24-hour fasting-induced food intake (K), normalized to body weight, measured over 24 hours in normal chow-fed MIC-1^fms and MIC-1^+/+ control mice at 25 weeks of age. L. Body weight of 25 week-old normal chow-fed MIC-1^fms and MIC-1^+/+ control mice before 24 hour fasting and at the indicated time points after re-introduction of food, with 100% representing pre-fasting body weight. M–O. Respiratory exchange ratio (RER, M), energy expenditure normalized to lean mass as determined by DXA (N) and ambulatory activity (O) of normal chow-fed MIC-1^fms and MIC-1^+/+ control mice at 26 weeks of age. Data are means ± SEM of 5 female mice per group. *p&lt;0.05, **p&lt;0.01 and ***p&lt;0.001 for the difference between genotypes.</p

<i>MIC-1/GDF15</i> expression in TRAMP and TRAMP^fmsmic-1 prostate and TC1-T5 cell line.

<p>(A) <i>MIC-1/GDF15</i> expression was quantified by qRT-PCR in the prostate of TRAMP and TRAMP^fmsmic-1 mice at 8, 17 and 25 weeks and normalized to <i>B-actin</i> expression as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043833#s2" target="_blank">material and methods</a>. (B) Relative <i>MIC-1/GDF15</i> expression was quantified by qRT-PCR in the TC1-T5 cell line (n = 3) and compared with TRAMP (n = 3) and TRAMP^fmsmic-1 (n = 3) prostate tumor after normalizing with <i>TBP</i> expression. Values are presented as mean normalized <i>MIC-1/GDF15</i> expression ±SEM. p values for the two-tailed unpaired <i>t</i> test are shown as ***p<i><</i>0.001.</p

Figure 3

<p><b>Female MIC-1^−/− mice eat more.</b> (A) Spontaneous 3 day cumulated food intake was measured in male and female MIC-1^−/− and control mice at 13 weeks of age. All mice were fed with standard chow diet ad libitum. Similar food intake was observed between male genotypes (<i>p = </i>0.3, <i>n = </i>8/group, t-test), female MIC-1^−/− mice had higher food intake relatively to the control mice (<i>p = </i>0.05, <i>n = </i>8/group). (B) Cumulated 24-hour fasting-induced food intake of was performed with the same group of mice at age of 14 weeks. MIC-1^−/− and control mice were fasted for 24 hours before re-introduction of food and spillage were collected at indicated time points, no genotypic difference were observed both male and female mice. Food intake at (C) light and (D) dark phase was also measured in the same group of mice at age of 12 weeks. No significant changes were observed between MIC-1^−/− and control mice in both sexes. Data are normalized to body weight plotted as means ± SE. Significance indicated as () for <i>p≤</i>0.05.</p

Figure 4

<p><b>Male MIC^−/− mice exhibit similar metabolic activity to their synergic control mice.</b> Metabolic activity of male MIC-1^−/− and control mice with groups of 16 at age between 14–16 weeks was determined by time course of (A) respiratory exchange rate (RER), (B) energy expenditure and (C) ambulatory activity. Energy expenditure was adjusted for lean mass via ANCOVA (common lean mass = 25.65 g). (D) Energy expenditure and (E) ambulatory activity were also presented as total for 24 hour, light phase and dark phase. Data are normalized to body weight and plotted as means ± SE.</p

Figure 5

<p><b>Female MIC^−/− mice exhibit lower metabolic activity than their synergic controls.</b> Metabolic activity of female MIC-1^−/− and control mice with groups of 9 at age between 14–16 weeks was determined by time course of (A) respiratory exchange rate (RER), (B) energy expenditure and (C) ambulatory activity. Energy expenditure (EE) was adjusted for lean mass via ANCOVA (common lean mass = 18.72 g), EE were significantly lower measured over 24 hour in MIC-1^−/− mice (<i>p = </i>0.001, <i>n = </i>9/group, <i>repeated measures ANOVA</i>). (D) MIC-1^−/− also displayed lower total EE in time courses over 24 hour, light phase and dark phase (<i>p = </i>0.001. <i>p = </i>0.005 and <i>p<</i>0.001, respectively, <i>n = </i>9/group, t-test). (E) Physical activity in dark phase were significantly lower in MIC-1^−/− mice (<i>p = </i>0.03, <i>n = </i>9, t-test). Data are normalized to body weight and plotted as means ± SE. Significance indicated as () for <i>p<</i>0.05 or () for <i>p<</i>0.01, or () for <i>p<</i>0.001.</p