FTO Protein Research Articles

Myocardial m6A regulators in postnatal development: effect of sex

N6-methyladenosine (m6A) is an abundant mRNA modification affecting mRNA stability and protein expression. It is a highly dynamic process, and its outcomes during postnatal heart development are poorly understood. Here we studied m6A machinery in the left ventricular (LV) myocardium of Fisher344 male and female rats (postnatal days one to ninety; P1-P90) using Western Blot. A downward pattern of target protein levels (demethylases FTO and ALKBH5, methyltransferase METTL3, reader YTHDF2) was revealed in male and female rat LVs during postnatal development. On P1, the FTO protein level was significantly higher in male LVs compared to females.

Investigation of the enhanced photoactivity of CdS/Bi2MoO6/MoSe2 and its application in antibody-free enzyme-assisted photoelectrochemical strategy for detection of N6-methyladenosine and FTO protein

N6-methyladenosine (m6A) is the most abundant and dynamic reversible modification of mRNA in eukaryotes discovered in the mid-1970s, which is widely involved in various physiological processes. Fat mass and obesity-associated protein (FTO protein) is not only a demethylase for m6A, but also a carcinogenic factor for many human cancers. Therefore, it is very important to build a simple and sensitive platform for simultaneous detection of m6A and FTO protein. To achieve this aim, a novel photoelectrochemical biosensor was constructed with the antibody-free detection strategy of DTT mediated –SH functionalization of m6A triggered by FTO protein catalyzed m6A oxidation. To achieve high detection sensitivity, CdS/Bi2MoO6/MoSe2 system with matched energy gap was employed as photoactive material. To achieve high detection selectivity, the –CH3 of m6A in the captured RNA sequence (m6A RNA) on the electrode surface was oxidized by FTO protein to form hydroxymethyl, followed with the covalent reaction of DTT with hydroxymethyl, making the labeling of m6A with –SH. Finally, a CdS nanoparticle was modified on the electrode through the specific covalent reaction between –SH and Cd, achieving the improvement of the photocurrent with the matched energy band. Under the optimal experimental conditions, the biosensor showed linear range from 0.001 to 50 nM and 0.0005–500 μg/L with the detection limit of 0.37 pM and 0.034 ng/L (S/N = 3) for m6A RNA and FTO protein, respectively. The applicability of the developed method was assessed by investigating the effect of entacapone on FTO protein activity.

E6E7 regulates the HK2 expression in cervical cancer via GSK3β/FTO signal

BackgroundCervical cancer is one of the most common cancers in women worldwide. Hexokinase 2 (HK2) is responsible for phosphorylating glucose into glucose-6-phosphate, which is required for tumorigenesis and metastasis. MethodsE6E7 and FTO were exogenously expressed, and their effects on HK2 mRNA and protein levels were detected by RT-qPCR and Western blot. ResultsThe exogenous expression of E6E7 in SiHa and C33A cells up-regulated the mRNA and protein levels of intracellular HK2, up-regulated the total m6A levels, changed the expression of m6A proteins and activated the GSK3β transcription. The expression levels of METTL3 and WTAP were enhanced, whereas the expression of FTO and ALKBH5 were decreased. In addition, FTO down-regulated the mRNA and protein levels of HK2. FTO overexpression partially inhibited the up-regulated expression of HK2 caused by E6E7. Furthermore, FTO overexpression increased the level of HK2 pre-mRNA in the nucleus and decreased the level of mature HK2 mRNA in the cytoplasm. We also found that GSK3β overexpression enhanced FTO ubiquitination and decreased FTO protein levels. ConclusionThis study found that E6E7 oncogene activates the transcription of GSK3β; GSK3β can promote the ubiquitination-proteasomal degradation of FTO and reduce the level of FTO protein; FTO inhibits the maturation and translation of HK2 mRNA by retaining HK2 pre-mRNA in the nucleus.

Investigation of FTO Gene-BMI Association in a Romanian Sample: a Functional Genomic Interpretation

Previously, we reported the results of an investigation regarding the FTO gene variant rs 1421085 association with BMI in a randomly selected Romanian sample, which showed a statistically relevant relationship. However, the small sample volume could not permit highlighting the hypothesized age factor role; the scarce literature information did not permit comments regarding the functional link between gene variation and BMI values. The actual work recapitulated the BMI-FTO association on an extended sample volume to 167, added the FTOrs17817449 variant, which has been reported in LD with the initial genotype, and approached a different statistical analysis (both of the separate and combined genotypes). The new results statistically confirmed the positive BMI-FTO association (p=0.018 and p=0.03 for each genotype rs1421085 and rs17817449, respectively p=0.03 for combined genotypes T/G and T/C, respectively); also, the age factor analysis showed the greater the age (after 40), the more tendency to gain weight (χ2=8.63). The observed high frequency of the risk rs1421805 genotype in our population and the age factor role in the FTO-BMI association are commented in the view of the newly emerged hypothesis regarding the climate influence on human migration during evolution and FTO polymorphic genotype distribution globally. Recent literature reports on the complex chromatin structure of the FTO locus, which environmental factors may influence, and on the FTO protein activity as an m6A demethylase are used in interpreting the biological role of this gene in adipose tissue formation, its maintenance, and lipolysis processes.

Solution structure ensemble of human obesity-associated protein FTO reveals druggable surface pockets at the interface between the N- and C-terminal domain

The fat mass and obesity-associated FTO protein catalyzes demethylation of the N6-methyladenosine, an epigenetic mark that controls several metabolic pathways by modulating the transcription, translation, and cellular localization of RNA molecules. Since the discovery that its overexpression links to the development of obesity and cancer, FTO was the target of screening campaigns and structure-based drug design efforts. Although several FTO inhibitors were generated, these often lack potency or selectivity. Herein, we investigate the structure and dynamics of human FTO in solution. We show that the structure of the catalytic N-terminal domain is unstable in the absence of the C-terminal domain, which explains why the isolated N-terminal domain is incompetent for catalysis and suggests that the domain interaction represents a target for the development of specific inhibitors. Then, by using NMR relaxation measurements, we show that the interface between the FTO structural domains, the active site, and several peripheral loops undergo conformational dynamics on both the picosecond–nanosecond and microsecond–millisecond timescales. Consistent with this, we found that the backbone amide residual dipolar couplings measured for FTO in phage pf1 are inconsistent with the static crystal structure of the enzyme. Finally, we generated a conformational ensemble for apo FTO that satisfies the solution NMR data by combining the experimental residual dipolar couplings with accelerated molecular dynamics simulations. Altogether, the structural ensemble reported in this work provides an atomic-resolution model of apo FTO and reveals transient surface pockets at the domain interface that represent potential targets for the design of allosteric inhibitors.

Identification and Validation of N6-Methyladenosine-Related Biomarkers for Bladder Cancer: Implications for Immunotherapy.

N6-methyladenosine (m6A) has emerged as one of the most important modifications of RNA. Based on the expression of 23 different modes of m6A regulatory factors, we identified three different m6A modification patterns in bladder cancer. The effects of the three different modes of m6A modification on clinicopathological characteristics, immune cell infiltration levels and expression levels of immune checkpoint genes were comprehensively analyzed. In addition, the effects of different modes of m6A modification on the therapeutic efficacy of anti-PD-L1 immunotherapy (atezolizumab) are also discussed. Our results confirm that m6A methylation plays an important role in immune cell recruitment in the tumor microenvironment of bladder cancer, which influences the efficacy of anti-PD-L1 therapy for bladder cancer. We further confirmed the important role of FTO protein in the biological function of bladder cancer cells by performing in vitro experiments. FTO functions as an oncogene in bladder cancer cells, and upon FTO knockdown, the level of m6A enzyme activity in bladder cancer cells was significantly increased, apoptosis was increased, and cell proliferation and cell invasion were reduced. In addition, our study also confirmed that K216H and K216E are probably important targets for regulating FTO. We provide new insights into the regulatory pathways of the immune microenvironment and the methylation function of m6A in bladder cancer, which will help in designing novel diagnostic methods, prognostic tools, and therapeutic targets.

Analysis of coding variants in the human FTO gene from the gnomAD database.

Single nucleotide polymorphisms (SNPs) in the first intron of the FTO gene reported in 2007 continue to be the known variants with the greatest effect on adiposity in different human populations. Coding variants in the FTO gene, on the other hand, have been little explored, although data from complete sequencing of the exomes of various populations are available in public databases and provide an excellent opportunity to investigate potential functional variants in FTO. In this context, this study aimed to track nonsynonymous variants in the exons of the FTO gene in different population groups employing the gnomAD database and analyze the potential functional impact of these variants on the FTO protein using five publicly available pathogenicity prediction programs. The findings revealed 345 rare mutations, of which 321 are missense (93%), 19 are stop gained (5.6%) and five mutations are located in the splice region (1.4%). Of these, 134 (38.8%) were classified as pathogenic, 144 (41.7%) as benign and 67 (19.5%) as unknown. The available data, however, suggest that these variants are probably not associated with BMI and obesity, but instead, with other diseases. Functional studies are, therefore, required to identify the role of these variants in disease genesis.

M6A demethylation of cytidine deaminase APOBEC3B mRNA orchestrates arsenic-induced mutagenesis

The cytidine deaminase APOBEC3B (A3B) is an endogenous inducer of somatic mutations and causes chromosomal instability by converting cytosine to uracil in single-stranded DNA. Therefore, identification of factors and mechanisms that mediate A3B expression will be helpful for developing therapeutic approaches to decrease DNA mutagenesis. Arsenic (As) is one well-known mutagen and carcinogen, but the mechanisms by which it induces mutations have not been fully elucidated. Herein, we show that A3B is upregulated and required for As-induced DNA damage and mutagenesis. We found that As treatment causes a decrease of N6-methyladenosine (m6A) modification near the stop codon of A3B, consequently increasing the stability of A3B mRNA. We further reveal that the demethylase FTO is responsible for As-reduced m6A modification of A3B, leading to increased A3B expression and DNA mutation rates in a manner dependent on the m6A reader YTHDF2. Our in vivo data also confirm that A3B is a downstream target of FTO in As-exposed lung tissues. In addition, FTO protein is highly expressed and positively correlates with the protein levels of A3B in tumor samples from human non–small cell lung cancer patients. These findings indicate a previously unrecognized role of A3B in As-triggered somatic mutation and might open new avenues to reduce DNA mutagenesis by targeting the FTO/m6A axis.

MiR-155 regulates m6A level and cell progression by targeting FTO in clear cell renal cell carcinoma

Although FTO, as an eraser of N6-methyladenosine (m6A), plays context-dependent tumor-suppressive and oncogenic roles in various cancer type, underlying molecular events of its aberrant expression in cancers is complex and still poorly understood. Here we show that miR-155 directly targets FTO to negatively regulate its expression and increased m6A level in ccRCC. Combining bioinformatics analysis and luciferase reporter assays, we identified that miR-155 directly bound to the 3′UTR of FTO mRNA and reduced FTO protein levels in ccRCC cells. Moreover, cell function assays, xenografts assays and m6A dot blot assays revealed that overexpression of miR-155 enhanced tumor cell proliferation and global mRNA m6A level, while decreasing apoptosis in a FTO-dependent manner. Collectively, our data demonstrates the functional importance of miR-155 in regulating FTO expression and global mRNA m6A level, and provides profound insights into ccRCC tumorigenesis.

Calmodulin as Ca2+-Dependent Interactor of FTO Dioxygenase.

FTO is an N6-methyladenosine demethylase removing methyl groups from nucleic acids. Several studies indicate the creation of FTO complexes with other proteins. Here, we looked for regulatory proteins recognizing parts of the FTO dioxygenase region. In the Calmodulin (CaM) Target Database, we found the FTO C-domain potentially binding CaM, and we proved this finding experimentally. The interaction was Ca2+-dependent but independent on FTO phosphorylation. We found that FTO–CaM interaction essentially influences calcium-binding loops in CaM, indicating the presence of two peptide populations—exchanging as CaM alone and differently, suggesting that only one part of CaM interacts with FTO, and the other one reminds free. The modeling of FTO–CaM interaction showed its stable structure when the half of the CaM molecule saturated with Ca2+ interacts with the FTO C-domain, whereas the other part is disconnected. The presented data indicate calmodulin as a new FTO interactor and support engagement of the FTO protein in calcium signaling pathways.

Deregulation of N6-Methyladenosine RNA Modification and Its Erasers FTO/ALKBH5 among the Main Renal Cell Tumor Subtypes.

(1) Background: Methylation of N6-adenosine (m6A) is the most abundant messenger RNA (mRNA) modification in eukaryotes. We assessed the expression profiles of m6A regulatory proteins in renal cell carcinoma (RCC) and their clinical relevance, namely, as potential biomarkers. (2) Methods: In silico analysis of The Cancer Genome Atlas (TCGA) dataset was use for evaluating the expression of the m6A regulatory proteins among RCC subtypes and select the most promising candidates for further validation. ALKBH5 and FTO transcript and protein expression were evaluated in a series of primary RCC (n = 120) and 40 oncocytomas selected at IPO Porto. (3) Results: In silico analysis of TCGA dataset disclosed altered expression of the major m6A demethylases among RCC subtypes, particularly FTO and ALKBH5. Furthermore, decreased FTO mRNA levels associated with poor prognosis in ccRCC and pRCC. In IPO Porto’s cohort, FTO and ALKBH5 transcript levels discriminated ccRCC from oncocytomas. Furthermore, FTO and ALKBH5 immunoexpression differed among RCC subtypes, with higher expression levels found in ccRCC comparatively to the other RCC subtypes and oncocytomas. (4) Conclusion: We conclude that altered expression of m6A RNA demethylases is common in RCC and seems to be subtype specific. Specifically, FTO and ALKBH5 might constitute new candidate biomarkers for RCC patient management, aiding in differential diagnosis of renal masses and prognostication.

End-labeling-based electrochemical strategy for detection of adenine methylation in nucleic acid by differential pulse voltammetry.

Apromising electrochemical strategy for assay of N6-methyladenosine (m6A)/N6-methyladenine (6mA) in RNA/DNA is proposed. The key of this strategy is the end-labeling of nucleic acid, which makes it possible to detect methylation level in unknown sequence. Firstly, the end of m6A-RNA or 6mA-DNA was labeled with sulfhydryl group through T4 polynucleotide kinase (T4 PNK) and then directly assembled on agold nanoparticle-modified glassy carbon electrode (AuNPs/GCE). Secondly, methylation sites in RNA/DNA were specifically recognized by anti-m6A-antibody, and then, horseradish peroxidase-labeled goat anti-rabbit IgG (HRP-IgG) was further conjugated on the antibody. Thirdly, HRP-IgG catalyzed the hydroquinone oxidation reaction to generate amplified current signal which correlates with the amount of m6A/6mA in nucleic acid. This method showed a wide linear range from 0.0001 to 10 nM for m6A-RNA, 0.001 to 100 nM for 6mA-dsDNA, and 0.0001 to 10 nM for 6mA-ssDNA. The method was successfully applied to detection of m6A/6mA in RNA/DNA from HeLa cells and E. coli cells and validation of the decrease of m6A-RNA in HeLa cells after treatment with FTO protein.

Structural characteristics of small-molecule inhibitors targeting FTO demethylase.

Studies have shown that the FTO gene is closely related to obesity and weight gain in humans. FTO is an N6-methyladenosine demethylase and is linked to an increased risk of obesity and a variety of diseases, such as acute myeloid leukemia, type 2 diabetes, breast cancer, glioblastoma and cervical squamous cell carcinoma. In light of the significant role of FTO, the development of small-molecule inhibitors targeting the FTO protein provides not only a powerful tool for grasping the active site of FTO but also a theoretical basis for the design and synthesis of drugs targeting the FTO protein. This review focuses on the structural characteristics of FTO inhibitors and discusses the occurrence of obesity and cancercaused by FTO gene overexpression.

Effect of Posttranslational Modifications on the Structure and Activity of FTO Demethylase.

The FTO protein is involved in a wide range of physiological processes, including adipogenesis and osteogenesis. This two-domain protein belongs to the AlkB family of 2-oxoglutarate (2-OG)- and Fe(II)-dependent dioxygenases, displaying N6-methyladenosine (N6-meA) demethylase activity. The aim of the study was to characterize the relationships between the structure and activity of FTO. The effect of cofactors (Fe2+/Mn2+ and 2-OG), Ca2+ that do not bind at the catalytic site, and protein concentration on FTO properties expressed in either E. coli (ECFTO) or baculovirus (BESFTO) system were determined using biophysical methods (DSF, MST, SAXS) and biochemical techniques (size-exclusion chromatography, enzymatic assay). We found that BESFTO carries three phosphoserines (S184, S256, S260), while there were no such modifications in ECFTO. The S256D mutation mimicking the S256 phosphorylation moderately decreased FTO catalytic activity. In the presence of Ca2+, a slight stabilization of the FTO structure was observed, accompanied by a decrease in catalytic activity. Size exclusion chromatography and MST data confirmed the ability of FTO from both expression systems to form homodimers. The MST-determined dissociation constant of the FTO homodimer was consistent with their in vivo formation in human cells. Finally, a low-resolution structure of the FTO homodimer was built based on SAXS data.

Autophagy of the m6A mRNA demethylase FTO is impaired by low-level arsenic exposure to promote tumorigenesis

Here we show that FTO as an N6-methyladenosine (m6A) RNA demethylase is degraded by selective autophagy, which is impaired by low-level arsenic exposure to promote tumorigenesis. We found that in arsenic-associated human skin lesions, FTO is upregulated, while m6A RNA methylation is downregulated. In keratinocytes, chronic relevant low-level arsenic exposure upregulated FTO, downregulated m6A RNA methylation, and induced malignant transformation and tumorigenesis. FTO deletion inhibited arsenic-induced tumorigenesis. Moreover, in mice, epidermis-specific FTO deletion prevented skin tumorigenesis induced by arsenic and UVB irradiation. Targeting FTO genetically or pharmacologically inhibits the tumorigenicity of arsenic-transformed tumor cells. We identified NEDD4L as the m6A-modified gene target of FTO. Finally, arsenic stabilizes FTO protein through inhibiting p62-mediated selective autophagy. FTO upregulation can in turn inhibit autophagy, leading to a positive feedback loop to maintain FTO accumulation. Our study reveals FTO-mediated dysregulation of mRNA m6A methylation as an epitranscriptomic mechanism to promote arsenic tumorigenicity.

Kinase GSK3β functions as a suppressor in colorectal carcinoma through the FTO-mediated MZF1/c-Myc axis.

Colorectal carcinoma (CRC) poses heavy burden to human health and has an increasing incidence. Currently, the existing biomarkers for CRC bring about restrained clinical benefits. GSK3β is reported to be a novel therapeutic target for this disease but with undefined molecular mechanisms. Thus, we aimed to investigate the regulatory effect of GSK3β on CRC progression via FTO/MZF1/c‐Myc axis. Firstly, the expression patterns of GSK3β, FTO, MZF1 and c‐Myc were determined after sample collection. Lowly expressed GSK3β but highly expressed FTO, MZF1 and c‐Myc were found in CRC. After transfection of different overexpressed and interference plasmids, the underlying mechanisms concerning GSK3β in CRC cell functions were analysed. Additionally, the effect of GSK3β on FTO protein stability was assessed followed by detection of MZF1 m6A modification and MZF1‐FTO interaction. Mechanistically, GSK3β mediated ubiquitination of demethylase FTO to reduce FTO expression. Besides, GSK3β inhibited MZF1 expression by mediating FTO‐regulated m6A modification of MZF1 and then decreased the proto‐oncogene c‐Myc expression, thus hampering CRC cell proliferation. We also carried out in vivo experiment to verify the regulatory effect of GSK3β on CRC via FTO‐mediated MZF1/c‐Myc axis. It was found that GSK3β inhibited CRC growth in vivo which was reversed by overexpressing c‐Myc. Taken together, our findings indicate that GSK3β suppresses the progression of CRC through FTO‐regulated MZF1/c‐Myc axis, shedding light onto a new possible pathway by which GSK3β regulates CRC.

Hsa-let-7b-5p Inhibits Proliferation of Human Leukemia THP-1 Cells via FTO/m6A/MYC Signaling Pathway

To investigate the down-regulation effect of let-7b-5p on the expression of FTO in acute myeloid leukemia cell line THP-1 and inhibitory effect on THP-1 proliferation via m6A/MYC signaling pathway. The acute myeloid leukemia cell line THP-1 and the normal human peripheral blood mononuclear cells (PBMNC) were selected as subjects. The expression of let-7b-5p and FTO mRNA in those cells was detected by qPCR, further the expression of FTO protein in those cells was detected by Western blot. And, the luciferase reporter gene assay was used to verify the targeting effect of let-7b-5p on FTO. Finally, THP-1 cells were transfected respectively with let-7b-5p mimic, and PBMNC with let-7b-5p inhibitor, there after the C-MYC mRNA m6A enrichment level in transfected cells was analyzed by dot blot, the expression levels of let-7b-5p, FTO and c-MYC were assayed by RT-PCR, the expressions of FTO and c-MYC protein were verified by Western blot, and the proliferation level of cells after transfection was detected by MTT assay. Compared with PBMNC, the expression of let-7b-5p in THP-1 significantly decreased, while the expression of FTO was significantly increased (P<0.05). After transfection with let-7b-5p mimic combined with FTO 3'-UTR, the luciferase activity of transfected THP-1 cells significantly decreased, but the luciferase activity significantly increased after transfection with mutant 3'-UTR, which was significantly different from the negative control group(blank vector) (P<0.05). Let-7b-5p inhibitor down-regulated c-MYC mRNA m6A enrichment, and then up-regulated the expression of FTO in transfected PBMNC cells, the effects of which were significant (P<0.05). However, let-7b-5p mimic up-regulated c-MYC mRNA m6A enrichment level and down-regulated the expression of FTO in the transfected THP-1 cells, and the cell proliferation rate was significantly lower than that in the negative control group (blank vector) (P<0.05). Human acute myeloid leukemia cell line THP-1 low expresses the let-7b-5p, which regulates c-MYC expression through let-7b-5p-/FTO-/m6A axis and promotes the proliferation of leukemia cell line THP-1.

The effects of folic acid on RNA m6A methylation in hippocampus as well as learning and memory ability of rats with acute lead exposure

• Lead exposure increased the level of Fat mass and obesity-associated protein expression in brain tissue. • Lead exposure resulted in decreased N6- methyladenosine levels, learning and memory ability. • The level of m6A after the intervention of folic acid, and the obvious improvement in learning and memory ability. The heavy metal lead in the environment is a neurotoxin with very strong biological toxicity which can cause serious damage to the learning and memory ability of humans and animals, folic acid can be involved in biological process to prevent impaired learning and memory. however, the specific mechanism is not clear. Herein, in order to explore whether folic acid has a protective effect on learning ousing on the M6A of FTO/METTL3 pathway control and learning and memory ability by lead exposure. Forty 6-week-old male rats were randomly divided into four groups, which drinking water freely model to establish folic acid (0.4 mg/ kg ·bw/day) intervention model of acute lead (0.2% lead acetate solution) exposure, and the determination of lead in hippocampus and Serum by GFAAP and the expression of FTO and METTL3 protein was detected by immunofluorescence assay, the methylation level of m6A in hippocampus was detected by EpiQuik TM m6A RNA methylation quantitative. A Morris water maze was used to test the spatial learning and memory ability of animals by positioning navigation experiment and space exploration experiment. Result display: Comparing to the control group, in the hippocampus, the expression of FTO in the lead exposure group was significantly increased ( P < 0.05), while METTL3 and M6A were significantly reduced in the lead exposure group ( P < 0.05). The ability of learning and memory in folic acid group was significantly high than that in lead exposure group ( P < 0.01). Our data showed that folic acid can effectively protect the body's ability of learning and memory by enhancing the mechanism of RNA m6A.

High intensity exercise downregulates FTO mRNA expression during the early stages of recovery in young males and females

BackgroundPhysical exercise and activity status may modify the effect of the fat mass- and obesity-associated (FTO) genotype on body weight and obesity risk. To understand the interaction between FTO’s effect and physical activity, the present study investigated the effects of high and low intensity exercise on FTO mRNA and protein expression, and potential modifiers of exercise-induced changes in FTO in healthy-weighted individuals.MethodsTwenty-eight untrained males and females (25.4 ± 1.1 years; 73.1 ± 2.0 kg; 178.8 ± 1.4 cm; 39.0 ± 1.2 ml.kg.min− 1 VO2peak) were genotyped for the FTO rs9939609 (T > A) polymorphism and performed isocaloric (400 kcal) cycle ergometer exercise on two separate occasions at different intensities: 80% (High Intensity (HI)) and 40% (Low Intensity (LO)) VO2peak. Skeletal muscle biopsies (vastus lateralis) and blood samples were taken pre-exercise and following 10 and 90 mins passive recovery.ResultsFTO mRNA expression was significantly decreased after HI intensity exercise (p = 0.003). No differences in basal and post-exercise FTO protein expression were evident between FTO genotypes. Phosphorylated adenosine monophosphate-activated protein kinase (AMPK) and Akt substrate of 160 kDa (AS160) were significantly increased following HI intensity exercise (p < 0.05). Multivariate models of metabolomic data (orthogonal two partial least squares discriminant analysis (O2PLS-DA)) were unable to detect any significant metabolic differences between genotypes with either exercise trial (p > 0.05). However, skeletal muscle glucose accumulation at 10 mins following HI (p = 0.021) and LO (p = 0.033) intensity exercise was greater in AA genotypes compared to TT genotypes.ConclusionOur novel data provides preliminary evidence regarding the effects of exercise on FTO expression in skeletal muscle. Specifically, high intensity exercise downregulates expression of FTO mRNA and suggests that in addition to nutritional regulation, FTO could also be regulated by exercise.Trial registrationACTRN12612001230842. Registered 21 November 2012 – Prospectively registered, https://www.anzctr.org.au/

Intracellular and tissue specific expression of FTO protein in pig: changes with age, energy intake and metabolic status

Genome-wide association studies in the FTO gene have identified SNPs correlating with obesity and type 2 diabetes. In mice, lack of Fto function leads to intrauterine growth retardation and lean phenotype, whereas in human it is lethal. The aim of this study in a pig model was to determine the localization of the FTO protein in different tissues and cell compartments, in order to investigate potential targets of FTO action. To better understand physiological role of FTO protein, its expression was studied in pigs of different age, metabolic status and nutrition, using both microscopic methods and Western blot analysis. For the first time, FTO protein was found in vivo in the cytoplasm, of not all, but specific tissues and cells e.g. in the pancreatic β-cells. Abundant FTO protein expression was found in the cerebellum, salivary gland and kidney of adult pigs. No FTO protein expression was detected in blood, saliva, and bile, excluding its role in cell-to-cell communication. In the pancreas, FTO protein expression was positively associated with energy intake, whereas in the muscles it was strictly age-related. In IUGR piglets, FTO protein expression was much higher in the cerebellum and kidneys, as compared to normal birth body weight littermates. In conclusion, our data suggest that FTO protein may play a number of distinct, yet unknown intracellular functions due to its localization. Moreover, it may play a role in animal growth/development and metabolic state, although additional studies are necessary to clarify the detailed mechanism(s) of action.