FTO inhibition mitigates high-fat diet-induced metabolic disturbances and cognitive decline in SAMP8 mice

Abstract

This study investigated the effects of fat mass and obesity-associated (FTO) inhibition on cognitive function and metabolic parameters of senescence-accelerated mouse prone 8 (SAMP8) mice fed a high-fat diet (HFD). SAMP8 mice fed an HFD exhibited increased body weight, impaired glucose tolerance, and elevated serum leptin levels. In epididymal white adipose tissue (eWAT), pharmacological treatment with FB23, a well-established FTO inhibitor, increased leptin production and modulated genes involved in lipid metabolism (Cpt1a, Atgl, Hsl, Fas), oxidative stress (OS) (Bip, Edem), and inflammation (Mcp1, Tnfα). Expression of hepatic genes related to lipid metabolism (Cpt1a, Atgl, Mgl, Dgat2, Srebp, Plin2) and OS (catalase, Edem) were modulated by FB23, although hepatic steatosis remained unchanged. Remarkably, FB23 treatment increased m6A RNA methylation in the brain, accompanied by changes in N6-methyladenosine (m6A)-regulatory enzymes and modulation of neuroinflammatory markers (Il6, Mcp1, iNOS). FTO inhibition reduced the activity of matrix metalloproteases (Mmp2, Mmp9) and altered IGF1 signaling (Igf1, Pten). Notably, enhanced leptin signaling was observed through increased expression of immediate early genes (Arc, Fos) and the transcription factor Stat3. Improved synaptic plasticity was evident, as shown by increased levels of neurotrophic factors (Bdnf, Ngf) and restored neurite length and spine density. Consistent with these findings, behavioral tests demonstrated that FB23 treatment effectively rescued cognitive impairments in SAMP8 HFD mice. The novel object recognition test (NORT) and object location test (OLT) revealed that treated mice exhibited enhanced short- and long-term memory and spatial memory compared to the HFD control group. Additionally, the open field test showed a reduction in anxiety-like behavior after treatment with FB23. In conclusion, pharmacological FTO inhibition ameliorated HFD-induced metabolic disturbances and cognitive decline in SAMP8 mice. These results suggest that targeting FTO may be a promising therapeutic approach to counteract obesity-induced cognitive impairment and age-related neurodegeneration.

Keywords: Aging; Epigenetics; FTO; Metabolic disorders; Neurodegenerative disease; m6A.

Fig. 1

Experimental design scheme. 4-month-old SAMP8 male mice were fed a control diet or 45% kcal HFD for one month. GTT was performed before starting FB23 (3 mg/kg) or vehicle oral gavage treatment. After 4 weeks, behavioral tests were carried out. Molecular analysis was done in the brain, serum, liver, and white- and brown-adipose tissues

Fig. 2

Body weight and glucose intolerance analysis in SAMP8 HFD mice. Body weight gain is shown as a percentage normalized to the initial weight A per week B at the end, and C food intake before treatment. D Fasting blood glucose values in mg/dL, E Glucose Tolerance Test (GTT), and F Area Under the Curve (AUC) in SAMP8 mice and SAMP8 mice fed a high-fat diet (HFD). Values are expressed as mean ± SEM. Groups were compared by two-way ANOVA analysis followed by Sidak’s post-hoc test (A, C, E). Groups were compared using unpaired Student’s t-test (B, D, F). n = 5–12 per group; Body weight gain is shown as a percentage normalized to the initial weight G per week and H at the end, and I diet intake follow-up after the treatment. SAMP8 HFD FB23 group is presented after the treatment. Values are expressed as mean ± SEM. Groups were compared using a two-way ANOVA analysis followed by Sidak’s post-hoc test (G, I). Groups were compared by unpaired one-way ANOVA analysis followed by Tukey’s post-hoc test (H). n = 5–12 per group; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001

Fig. 3

Characterization of lipid metabolism in eWAT. A Representative eWAT gene expression of Leptin. B Serum leptin levels expressed in mg/mL. C Representative eWAT gene expression of Pparα, Cpt1α, Atgl, Hsl and Fas, D Sod, Catalase, Bip, Edem and Pdi, and E Mcp1, Il-6, Il-1β and Tnfα. Relative mRNA expression levels were determined by real-time PCR. Values are expressed as mean ± SEM. Groups were compared by one-way ANOVA analysis followed by Tukey’s post-hoc test. If necessary, groups were compared using a two-tailed Student’s t-test. n = 3–4 per group. *p < 0.05; **p < 0.01

Fig. 4

Characterization of lipid and glucose metabolism in liver. Hepatic A triglycerides (TG) content and B cholesterol content (CHOL) in mg/g liver tissue. Relative liver gene expression of C Pepck, Cpt1α, Atgl and Mgl, D Dgat2, Srebp and Plin2, and E Sod, Catalase, Bip, Edem and Pdi. Relative mRNA expression levels were determined by real-time PCR. Values are expressed as mean ± SEM. Groups were compared by one-way ANOVA analysis followed by Tukey’s post-hoc test. If necessary, groups were compared using a two-tailed Student’s t-test. n = 3–4 per group. *p < 0.05; **p < 0.01

Fig. 5

Minor changes in BAT metabolic function. Representative BAT gene expression of A Ucp1, Cidea, Bmp8b, Pparγ, Prdm16, Pgc1α, Glut4 and Cpt1b, and B Sod, Catalase, Bip, Edem and Pdi. Relative mRNA expression levels were determined by real-time PCR. Values are expressed as mean ± SEM. Groups were compared by one-way ANOVA analysis followed by Tukey’s post-hoc test. n = 3–4 per group. *p < 0.05

Fig. 6

Modulation in m6A methylation and neuroinflammatory markers in SAMP8 HFD mice after FB23 treatment. A Quantification of m6A RNA methylation levels in the brain. OD values were determined using the EpiQuik™ m6A RNA methylation Quantification Kit. Representative gene expression in the brain of B m6A-regulatory enzymes: Fto, Ythdc1, Ythdc2, Ythdf1, Ythdf2 and Ythdf3, C Il-6, D Mcp1, E iNos, F Chop, G Atf3, H Atf6, I Bip and J Catalase. Relative mRNA expression levels were determined by real-time PCR. Values are expressed as mean ± SEM. Groups were compared by one-way ANOVA analysis followed by Tukey’s post-hoc test. n = 3–4 per group (outliers: n = 1 in the SAMP8 HFD group for Mcp1 ratio values and n = 1 in the SAMP8 group for Atf6 ratio values); *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001

Fig. 7

Decrease in IGF1/AKT activation pathway in SAMP8 HFD mice after FB23 treatment. Representative gene expression in the brain of A Igf1, B Pi3k and C Pten. Immunoblot and quantification of D, E p-AKT/AKT ratio levels and F, G p-NFKB/NFKB ratio levels. Representative gene expression of (H) Cxcl10, (I) p53, (J) Mmp-2 and (K) Mmp-9. Relative mRNA expression levels were determined by real-time PCR. Protein ratio levels were determined by Western blotting. Values are expressed as mean ± SEM. Groups were compared by one-way ANOVA analysis followed by Tukey’s post-hoc test. n = 3–4 per group (outliers: n = 1 in the SAMP8 group for Pten gene levels; n = 1 in the SAMP8 HFD FB23 group for p-AKT/AKT protein levels; n = 1 in the SAMP8 HFD group for p53 gene levels); *p < 0.05; **p < 0.01

Fig. 8

Increased synaptic plasticity in the cortical tissue of SAMP8 HFD mice after FB23 treatment. Representative gene expression in the brain of A Leptin receptor, B Arc, C cFos, D Stat3, E Bdnf, F Ngf, G Vgf and H Scg2. The relative mRNA expression levels were determined by real-time PCR. Values are expressed as mean ± SEM. Groups were compared by one-way ANOVA analysis followed using a Tukey’s post-hoc test. n = 3–4 per group; *p < 0.05; **p < 0.01. I Representative images and tracings of Golgi-stained cortical neurons (scale bar = 100 µm) and J quantification of the number of neuronal intersections vs. distance from the soma. K Representative images of dendritic spine density (scale bar = 5 µm) by Golgi staining and L quantification. Values are expressed as mean ± SEM. Groups were compared using one-way ANOVA analysis followed by Tukey’s post-hoc test. n = 100 per group for neurons and spines (I, K); ****p < 0.0001

Fig. 9

Rescued cognitive impairment in SAMP8 HFD mice after FB23 treatment. Results of the behavioral and cognitive tests and analysis. The time spent in the center (A) and periphery (B) was measured by OFT and expressed as a percentage. The number of C rearing, D grooming, E defecation and F urination were also counted. G Familiarization phase and H test of spatial memory with OLT. I Familiarization phase and J short-term memory test after 2 h and K long-term memory after 24 h with NORT. The discrimination index (DI) was determined for both tests. The values are given as mean ± SEM. Groups were compared using one-way ANOVA analysis followed by Tukey’s post-hoc test. n = 5–6 per group; *p < 0.05; **p < 0.01; ***p < 0.001

Fig. 10

FTO inhibition ameliorates metabolic dysregulation and cognitive decline in SAMP8 mouse model after HFD