A link between FTO, ghrelin, and impaired brain food-cue responsivity

Abstract

Polymorphisms in the fat mass and obesity-associated gene (FTO) are associated with human obesity and obesity-prone behaviors, including increased food intake and a preference for energy-dense foods. FTO demethylates N6-methyladenosine, a potential regulatory RNA modification, but the mechanisms by which FTO predisposes humans to obesity remain unclear. In adiposity-matched, normal-weight humans, we showed that subjects homozygous for the FTO "obesity-risk" rs9939609 A allele have dysregulated circulating levels of the orexigenic hormone acyl-ghrelin and attenuated postprandial appetite reduction. Using functional MRI (fMRI) in normal-weight AA and TT humans, we found that the FTO genotype modulates the neural responses to food images in homeostatic and brain reward regions. Furthermore, AA and TT subjects exhibited divergent neural responsiveness to circulating acyl-ghrelin within brain regions that regulate appetite, reward processing, and incentive motivation. In cell models, FTO overexpression reduced ghrelin mRNA N6-methyladenosine methylation, concomitantly increasing ghrelin mRNA and peptide levels. Furthermore, peripheral blood cells from AA human subjects exhibited increased FTO mRNA, reduced ghrelin mRNA N6-methyladenosine methylation, and increased ghrelin mRNA abundance compared with TT subjects. Our findings show that FTO regulates ghrelin, a key mediator of ingestive behavior, and offer insight into how FTO obesity-risk alleles predispose to increased energy intake and obesity in humans.

Figures

Figure 1. Hunger scores, plasma acyl-ghrelin levels, and appeal of hedonic images in TT and AA subjects.

(A) Standard test meal study protocol. On the day prior to the study, subjects consumed the same evening meal at 20:00, then fasted and drank only water. On the study day, a venous cannula was inserted into a left forearm vein, and a period of 1 hour was allowed for acclimatization. At t0, a baseline blood sample and appetite VAS were taken. Subjects were then given a standard test meal to consume within 20 minutes. Blood samples and appetite VAS were taken at t20 and t30 and every 30 minutes thereafter until t180. (B–D) Postprandial responses in 10 TT (blue squares) and 10 AA (red circles) adiposity-matched subjects to a meal at t0. (B) Δ Hunger, (C) Δ plasma acyl-ghrelin concentrations, and (D) AUC Δ acyl-ghrelin in TT (blue bar) and AA (red bar) subjects. (E–G) fMRI fed–study day postprandial responses in 12 TT (blue squares) and 12 AA (red circles) adiposity-matched subjects to a meal at t0. (E) Δ Hunger, (F) Δ plasma acyl-ghrelin concentrations, and (G) AUC Δ acyl-ghrelin suppression in TT (blue bar) and AA (red bar) subjects. (H) Postprandial appeal ratings of hedonic, high-calorie food images from 12 TT (blue bar) and 12 AA (red bar) fMRI study subjects. (I and J) Combined postprandial (I) AUC Δ hunger reduction and (J) AUC Δ acyl-ghrelin suppression in 22 TT (blue bars) and 22 AA (red bars) subjects. Data are presented as the mean ± SEM. *P < 0.05; **P < 0.01.

Figure 2. Effect of the FTO rs9939609 genotype on BOLD responses to food and non-food images.

(A–F) Axial slices with superimposed group functional activity. (A–C) Brain regions where the TT and AA genotypes exhibited significantly different BOLD responses while viewing food images (all food greater than non-food) in the fasted state, with TT subjects showing a greater BOLD response than AA subjects. (D–F) Brain regions where a marked interaction between the FTO genotype (TT versus AA) and nutritional state (fed versus fasted) was found in BOLD responses to high-incentive- versus low-incentive-value food images (high-calorie greater than low-calorie). Left side of each panel is the left side of the brain. z is the MNI space z coordinate of the axial slice. T color scale reflects the T value of the functional activity. Results are presented at a threshold of P < 0.05, FWE corrected on the basis of cluster extent. Ant. insula, anterior insula. (G) Mean response (β coefficients) for the left anterior insula/OFC (BA47) cluster displaying genotype-by-nutritional state interaction for the high-calorie versus low-calorie contrast. Graph shows cluster mean parameter estimates ± SEM for high-calorie (hatched bars) and low-calorie food images (bars without hatching) in the fasted (open bars) and fed (solid bars) states in TT (blue bars) and AA (red bars) subjects. HC, high-calorie; LC, low-calorie.

Figure 3. Modulatory effects of acyl-ghrelin on BOLD responses in the fasted state.

(A–D) Axial slices with superimposed group functional activity. Brain regions where TT and AA subjects significantly differed in their relationship between BOLD response to high-incentive- versus low-incentive-value food images (high-calorie greater than low-calorie) and circulating acyl-ghrelin in the fasted state. The left side of each panel is the left side of the brain. z is the MNI space z coordinate of the axial slice. T color scale reflects the T value of the functional activity. Results are presented at a threshold of P < 0.05, FWE corrected on the basis of cluster extent. (E–H) Regression plots between the BOLD response to high-calorie versus low-calorie food images and circulating acyl-ghrelin in the TT (blue, open squares) and AA (red, open circles) groups in the fasted state. A positive relationship (E and G) was found in the TT group, whereas a negative regression coefficient (F and H) was seen in the AA group. (E and F) The plotted β coefficients were extracted from the cluster peak within the hypothalamus (MNI space x, y, and z coordinates for the peak cluster voxel [8, –4, –4]). (G and H) Plotted β coefficients were extracted from the cluster peak within the nucleus accumbens (NA) (MNI space x, y, and z coordinates for the peak cluster voxel [6, –6, –4]).

Figure 4. Modulatory effects of acyl-ghrelin on BOLD responses in the fed state.

(A–D and G) Axial slices with superimposed group activity. MOG, middle occipital gyrus. (A–D) Brain regions where the TT and AA groups significantly differed in their relationship between food-related (all food greater than non-food) BOLD response and postprandial circulating acyl-ghrelin suppression (t0–t54). P < 0.05, FWE corrected. The left side of each panel is the left side of the brain. z is the MNI space z coordinate of the axial slice. T color scale reflects the T score of the interaction. (E and F) Regression plots between food-related BOLD response and circulating postprandial acyl-ghrelin suppression (t0–t54) in TT (blue, open squares) and AA (red, open circles) subjects. Positive regression and negative coefficients (β) were found in the TT (E) and AA (F) groups, respectively. Plotted coefficients were extracted from the cluster peak within the left cuneus (MNI space x, y, and z coordinates for the peak cluster voxel cluster [–12, –92, 28]). (G) Right caudate nucleus where the TT and AA groups exhibited a divergent relationship between BOLD response to hedonic food images and postprandial circulating acyl-ghrelin suppression (t0–t54). (H and I) Regression plots between BOLD response to hedonic food images and circulating postprandial ghrelin suppression in TT (blue, open squares) and AA (red, open circles) subjects. Negative and positive βs were found in the TT (H) and AA (I) groups, respectively. Plotted coefficients were extracted from the cluster peak within the right caudate nucleus (MNI space x, y, and z coordinates for the peak cluster voxel [22, 8, 18].

Figure 5. Effect of FTO overexpression on ghrelin mRNA abundance, total ghrelin, acyl-ghrelin, and GOAT mRNA abundance in MGN3-1 and HEK293T cells.

(A–E) Effect of FTO overexpression (red bars) compared with the control vector (blue bars) in MGN3-1 cells on (A) Ghrl mRNA abundance, (B) total ghrelin, (C) acyl-ghrelin, (D) acyl-ghrelin/total ghrelin, and (E) Goat mRNA abundance. (F–J) Effect of FTO overexpression (red bars) compared with control vector (blue bars) in HEK293T cells. (F) GHRL mRNA abundance, (G) GOAT mRNA abundance, (H) total ghrelin, (I) acyl-ghrelin, and (J) acyl-ghrelin/total ghrelin. Data are presented as the mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 6. Effect of FTO overexpression on immunodepletion of m6A-methylation of ghrelin mRNA and Rps14 mRNA from total cellular mRNA in (A and C) MGN3-1 cells and (B and D) HEK293T cells.

mRNA abundance was determined by qPCR and calculated relative to the input sample, with transcripts normalized to the amount of Gusb mRNA (a non-m6A–containing mRNA). n = 4 per study group, and results are representative of 2 independent experiments. Data are the mean ± SEM. Differences between normalized control unbound and normalized FTO unbound; ##P < 0.01; ###P < 0.001.

Figure 7. Effect of the rs9939609 FTO genotype on GHRL mRNA abundance, immunodepletion of m6A-methylated GHRL mRNA, and RPS14 mRNA in peripheral blood cells.

(A) GHRL mRNA abundance in peripheral blood cells from TT (blue bars) and AA (red bars) study subjects. Effect of the FTO genotype on immunodepletion of m6A-methylated (B) GHRL mRNA and (C) RPS14 mRNA abundance from total cellular mRNA in TT and AA study subjects. mRNA abundance was determined by qPCR and calculated relative to the input sample, with transcripts normalized to the amount of GUSB mRNA (a non-m6A–containing mRNA). (D) FTO mRNA abundance in peripheral blood cells. Data are presented as the mean ± SEM; n = 10 per genotype. **P < 0.01. Difference between normalized TT unbound and normalized AA unbound; ###P < 0.001.