Overexpression of Fto leads to increased food intake and results in obesity

Abstract

Genome-wide association studies have identified SNPs within FTO, the human fat mass and obesity-associated gene, that are strongly associated with obesity. Individuals homozygous for the at-risk rs9939609 A allele weigh, on average, ~3 kg more than individuals with the low-risk T allele. Mice that lack FTO function and/or Fto expression display increased energy expenditure and a lean phenotype. We show here that ubiquitous overexpression of Fto leads to a dose-dependent increase in body and fat mass, irrespective of whether mice are fed a standard or a high-fat diet. Our results suggest that increased body mass results primarily from increased food intake. Mice with increased Fto expression on a high-fat diet develop glucose intolerance. This study provides the first direct evidence that increased Fto expression causes obesity in mice.

Figure 1. Generation of a mouse model overexpressing Fto

(a) Relative Fto expression in the indicated tissues of FTO-2 (n=10) FTO-3 (n=10) and FTO-4 (n=10) mice. Epi, epigonadal. Ab, abdominal. Sub, subcutaneous. WAT, white adipose tissue. Data are expressed as mean±standard error (s.e.m.). *P<0.05, **P<0.01, ***P<0.0001. ns, non-significant. (b) Representative Western blots of FTO and actin (loading control) from skeletal muscle and liver from FTO-2, FTO-3 and FTO-4 mice. (c) RT-PCR of EGFP using brain cDNA prepared from FTO-2, FTO-3 and FTO-4 mice. Hypoxanthine-guanine phosphoribosyltransferase (Hprt1) is included as a control.

Figure 2. Fto dose-dependent increases in body weight are observed in male and female mice on standard (SD) and high fat (HFD) diets

(a) Females SD, FTO-2 (wild-type, n=16), FTO-3 (n=28, P=0.0003) and FTO-4 (n=16, P<0.0001) (b) Females HFD, FTO-2 (n=15), FTO-3 (n=14, P=0.04) and FTO-4 (n=15, P=0.002) (c) Males SD, FTO-2 (n=16), FTO-3 (n=31, P=0.001) and FTO-4 (n=16, P=0.0001). (d) Males HFD, FTO-2 (n=18), FTO-3 (n=15, P=0.01) and FTO-4 (n=16, P<0.0001). Data are expressed as mean±s.e.m. Statistical analysis was performed using a repeated measures ANOVA. All P values are against FTO-2.

Figure 3. Body Composition varies with Fto copy number

(a) Body weight of 20-week old male and female mice on a standard (SD) and high fat diet (HFD). Males: SD, FTO-2 (n=16), FTO-3 (n=31), FTO-4 (n=15); HFD, FTO-2 (n=18), FTO-3 (n=15), FTO-4 (n=16). Females: SD, FTO-2 (n=16), FTO-3 (n=28), FTO-4 (n=16); HFD, FTO-2 (n=15), FTO-3 (n=14), FTO-4 (n=15). (b) Total fat mass measured by DEXA scanning in male and female mice on standard (SD) and high fat diets (HFD). Males: SD, FTO-2 (n=16), FTO-3 (n=30), FTO-4 (n=15); HFD, FTO-2 (n=17), FTO-3 (n=15), FTO-4 (n=16). Females: SD, FTO-2 (n=16), FTO-3 (n=28), FTO-4 (n=16); HFD, FTO-2 (n=16), FTO-3 (n=14), FTO-4 (n=15). (c,d) Weights of epigonadal WAT (c) and abdominal WAT (d) in mice overexpressing Fto. Males: SD, FTO-2 (n=16), FTO-3 (n=21), FTO-4 (n=15); HFD, FTO-2 (n=15), FTO-3 (n=14), FTO-4 (n=14). Females: SD, FTO-2 (n=15), FTO-3 (n=28), FTO-4 (n=15); HFD, FTO-2 (n=12), FTO-3 (n=14), FTO-4 (n=14). (e) Epigonadal adipocyte area is increased in female mice on both standard and high fat diets and in males on a HFD (n=5 in each case). (f) Lean body mass in male and female mice on SD and HFD. Same mouse numbers as in (b). Data (a-f) are expressed as mean±s.e.m. *P<0.05, **P<0.01, ***P<0.0001.

Figure 4. Effects of FTO on energy intake and plasma leptin

(a) Food intake over 24 hours normalised to body weight (BW), measured in 10 and 19-week old mice. Males: SD, FTO-2 (n=16) FTO-3 (n=31) and FTO-4 (n=16); HFD, FTO-2 (n=18) FTO-3 (n=15) and FTO-4 (n=16). Females: SD, FTO-2 (n=16), FTO-3 (n=28) and FTO-4 (n=16); HFD, FTO-2 (n=15) FTO-3 (n=14) and FTO-4 (n=15). (b) Plasma leptin levels at 8 weeks of age adjusted for body weight (BW) following an overnight 16-hour fast. Males: SD, FTO-2 (n=14), FTO-3 (n=25), FTO-4 (n=14); HFD, FTO-2 (n=12), FTO-3 (n=12), FTO-4 (n=12). Females: SD, FTO-2 (n=16), FTO-3 (n=25), FTO-4 (n=14); HFD, FTO-2 (n=13), FTO-3 (n=10), FTO-4 (n=10). (c) Relative Leptin (Lep) gene expression in 20 week-old female epigonadal (Epi), abdominal (Ab) and subcutaneous (sub) WAT. FTO-2 (n=10), FTO-3 (n=10), FTO-4 (n=10). (d) Relative gene expression of hypothalamic neuropeptides. FTO-2 (n=10) FTO-3 (n=10) and FTO-4 (n=10) mice. Data (a-d) are expressed as mean±s.e.m. *P<0.05, **P<0.01, ***P<0.0001.

Figure 5. Effects of Fto on energy expenditure and physical activity

(a) Male and (b) Female heat production over a 22-hour period during the light and dark phases for 18-week old male (a,b) and female (c,d) mice on a standard (SD) or high fat diet (HFD). Males: SD, FTO-2 (n=15), FTO-3 (n=25), FTO-4 (n=16); HFD, FTO-2 (n=12), FTO-3 (n=12), FTO-4 (n=15). Females: SD, FTO-2 (n=16), FTO-3 (n=22), FTO-4 (n=15); HFD, FTO-2 (n=12), FTO-3 (n=13), FTO-4 (n=14). (c) Physical activity measured as the number of rotations of an activity wheel in a 7-day period, following a 3-day entrainment period. Males and females: FTO-2 (n=7), FTO-3 (n=7), FTO-4 (n=6). Data (a-c) are expressed as mean±s.e.m. *P<0.05, **P<0.01

Figure 6. Glucose homeostasis and Fto overexpression

(a) Area under the curve (AUC) during a 120-minute IPGTT in 12-week old mice. Males: SD, FTO-2 (n=15), FTO-3 (n=25), FTO-4 (n=16); HFD, FTO-2 (n=12), FTO-3 (n=13), FTO-4 (n=12). Females: SD, FTO-2 (n=16), FTO-3 (n=22), FTO-4 (n=15); HFD, FTO-2 (n=12), FTO-3 (n=12), FTO-4 (n=13). (b) AUC analysis for glucose during a 30-minute IPGTT in 16-week old mice. Males: SD, FTO-2 (n=15), FTO-3 (n=23), FTO-4 (n=15); HFD, FTO-2 (n=12), FTO-3 (n=12), FTO-4 (n=14). Females: SD, FTO-2 (n=16), FTO-3 (n=22), FTO-4 (n=15); HFD, FTO-2 (n=12), FTO-3 (n=11), FTO-4 (n=15). (c) Adiponectin levels at 20-weeks of age following a 6-hour light phase fast. Males: SD, FTO-2 (n=12), FTO-3 (n=22), FTO-4 (n=10); HFD, FTO-2 (n=12), FTO-3 (n=10), FTO-4 (n=10). Females: SD, FTO-2 (n=11), FTO-3 (n=22), FTO-4 (n=9); HFD, FTO-2 (n=10), FTO-3 (n=9), FTO-4 (n=10). Data (a-c) are expressed as mean±s.e.m. *P<0.05, **P<0.01, ***P<0.0001.