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. Author manuscript; available in PMC: 2025 Mar 14.
Published in final edited form as: Biochem J. 2012 Jun 1;444(2):183–7. doi: 10.1042/BJ20120065

Kinetic analysis of FTO (Fat mass and obesity related) reveals that it is unlikely to function as a sensor for 2-oxoglutarate

Marcella Ma 1, Heather P Harding 1, Stephen O’Rahilly 1, David Ron 1, Giles SH Yeo 1,✉
PMCID: PMC7617487  EMSID: EMS203458  PMID: 22435707

Abstract

Genomewide-association studies have revealed that SNPs in FTO (Fat mass and obesity-associated transcript) are robustly associated with BMI and obesity. FTO is an Fe(II) 2-oxoglutarate (2OG) dependent dioxygenase that can demethylate 3-methylthymine (3-meT) in single-stranded DNA, as well as 3-methyluracil (3-meU) and N6-methyl adenosine in RNA. Here, we describe the development of an RNase cleavage assay measuring FTO’s demethylation activity on 3-methyluracil. RNase A cleaves at the 3’end of pyrimidines, including uracil, and a methyl group at position three of uracil inhibits cleavage. An oligonucleotide probe was designed consisting of a DNA stem, a RNA loop containing a single 3meU as the only RNase A cleavage site, a fluorescent reporter on one end and a quencher at the other. FTO demethylation of the unique 3-methyluracil enables RNase A cleavage, releasing the quencher and enabling a fluorescent signal. In the presence of excess RNase A, FTO activity is limiting to the development of fluorescent signal, which can be read continuously and is able to discriminate between wildtype and the catalytically dead R316Q FTO. 2-oxoglutarate is a co-substrate of FTO and as a metabolite in the citric-acid cycle is a marker of intracellular nutritional status. Our assay was used to measure, for the first time, the Km of FTO for 2-oxoglutarate. The Km of 2.88uM is up to 10-fold lower than the estimated intracellular concentrations of 2-oxoglutarate, rendering it unlikely that FTO functions as a sensor for 2-oxoglutarate levels.

Introduction

In 2007, Genomewide association studies revealed that single nucleotide polymorphisms (SNPs) within intron 1 of FTO (Fat mass and obesity related transcript) are robustly associated with body mass index and obesity [1]. Many subsequent studies, covering more than 22 distinct populations of European, African and Asian ancestries, have confirmed the association of FTO with BMI (reviewed by [2]). It is clear from the weight of evidence that the major effect of SNPs in FTO is on increased energy intake, with reduction in satiety.

FTO is widely expressed across multiple tissues, although it is most highly expressed in the brain especially in the hypothalamus, a region that plays a key role in the control of energy homeostasis [3]. We have found that expression of FTO, specifically in the arcuate nucleus (ARC) of the hypothalamus, is bi-directionally regulated as a function of nutritional status; decreasing following a 48hr fast and increasing after 10 weeks of exposure to a high fat diet, and that modulating FTO levels specifically in the ARC can influence food intake [3, 4]. FTO shares sequence motifs with Fe(II)- and 2-oxoglutarate (2-OG) dependent oxygenases. In vitro, recombinant FTO is able to catalyze the Fe(II)- and 2OG-dependent demethylation of 3-methylthymine (3-meT) in single-stranded DNA [3], as well as 3-methyluracil (3-meU) [5] and 6-methyl-adenosine [6] in single-stranded RNA, with concomitant production of succinate, formaldehyde and carbon dioxide, suggesting a potential role for FTO in nucleic acid repair or modification. Its selectivity for single-stranded over double-stranded nucleic acids, which could be predicted from its crystal structure [7] and has also been demonstrated empirically [5], suggests that RNA would be its prime substrate. Because 2-OG, a key intermediate in the citric-acid cycle, is a co-substrate of FTO, it is possible that FTO functions as a sensor for intracellular concentrations of this metabolite and thus cellular metabolism. However, the sensitivity of FTO to the concentration of 2-OG has not been measured and it remains unknown if FTO activity is likely to be modulated by physiological variation in its substrate’s concentration.

The SNP-bearing obesity-associated alleles of FTO are highly prevalent, thus altered FTO function has the potential to impact the bodyweight of up to 1 billion people. This has engendered considerable interest in the biochemical activity of FTO and has raised the spectre of modifying this activity for therapeutic purpose. Current assays of FTO function are all endpoint assays that rely on radioactive tracers or HPLC detection of substrate and products [3, 5, 6, 8]. They require large amounts of purified FTO enzyme, are time-consuming and of limited utility in measuring enzyme kinetics. We set out to develop a microlitre-scale, continuous, homogenous, fluorescence based assay, suited to analysis of enzyme kinetics and high-throughput screens and applied our assay to examine the enzyme kinetics of FTO with regards to its usage of 2-OG.

Experimental

Recombinant wild type and mutant FTO purification

Recombinant wild type and mutant FTO proteins were purified as previously described [8, 9]. Briefly, expression plasmids were transformed into E. coli BL21-Gold (DE3) (Stratagene) and cultured in LB broth and 50 μg/ml carbenicillin to A600 1.0 at 37°C. Expression of the cloned gene was induced by addition of 0.5 mM isopropyl-β-D-1-thiogalactopyranoside (IPTG) at 15°C for 4h. The cells were harvested and pellets were stored at -80°C until day of the protein purification. Cell pellets were resuspended in lysis buffer (50 mM HEPES-KOH pH8.0, 2 mM β-mercaptoethanol, 5% glycerol and 300 mM NaCl) before digestion with lysozyme (1 mg/ml). Followed by sonication, the lysate was collected after centrifugation at 15,000 x g for 30 minutes at 4°C. The cleared lysate was supplemented with imidazole final at 10 mM before mixing with 1 ml of pre-washed Ni-NTA beads (Qiagen). After binding for an hour in cold, the mixture was re-loaded to the column and washed 3 times with 10 ml each of lysis buffer supplemented with 10 mM imidazole, followed by 7.5 ml lysis buffer supplemented with 40 mM imidazole. FTO was finally eluted with 2 ml lysis buffer containing 250 mM imidazole. The eluate was concentrated with a 30 kDa MWCO concentrator (Sartorius Stedim; Epsom, UK) with buffer changing to 20 mM HEPES-KOH pH8, 5% glycerol and 50 mM NaCl. Purified proteins were snap frozen and stored at -80°C. Protein purity was estimated by Commassie stain after resolving in 4-12% SDS-PAGE (Invitrogen).

FTO 3meU demethylation assay

The stem-loop substrate containing a methylated uridine at the N3 position was manufactured by Midland Certified Reagent Co. (Midland, USA). Upon arrival, the substrate was re-suspended in nuclease-free water to a concentration of 100 μM and stored at -80°C. An optimal RNase A concentration was determined before assaying FTO demethylation activity. The florescent FTO demethylation activity assay was modified from our assay using 14C-3-methyl-thymidine labelled DNA substrate as previously reported [8, 9]. After initial optimization testing any interference on the florescence signal from components, in each reaction, 100 nM of either methylated or unmethylated substrate was mixed with 75 μM Fe(NH4)2(SO4)2, 300 μM 2-oxoglutarate, 2 mM ascorbate, 50 μg/ml bovine serum albumin (BSA) and various amount of RNase A from 10-12 μg/μl to 0.1 μg/μl in 50 mM Tris buffer at pH7.0. Samples were prepared in duplicates and the FAM emission was measured for 30 min at a wavelength of either 520 nm or 535 nm (depending on the plate-reader) with excitation at 485 nm. The measurement was performed using a microplate reader (BMG Pherastar (520 nm), Labtech, Germany or Infinite f500 (535 nm); Tecan, Switzerland) in a dark flat bottom 96-well plate at room temperature.

Similar to the previous set up, 100 nM of the methylated substrate was incubated with the reaction mix with 625 pg of RNase A per reaction, Various FTO was added from 0 to 1000nM. Samples were prepared in duplicates and the FAM emission was measured for 30 min at a wavelength of 535 nm with excitation at 485 nm. The measurement was performed using a Tecan Infinite f500 microplate reader in a dark flat bottom 96-well plate at room temperature.

Data and statistical analyses

The experiments in Fig 1b and Fig 2b were each performed using one separate preparation of FTO. Experiments in Fig 2a, 3 and 4 are representative plots, with the each experiment performed twice, using two different preparations of FTO. All data calculations and figures were performed using Microsoft Excel and GraphPad Prism.

Figure 1. Principle and verification of the RNase A coupled FTO demethylase assay.

Figure 1

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a. The oligonucleotide has a stem-loop structure where the stem is formed from DNA (to prevent cleavage by RNase A) and the loop from RNA, with the sole pyrimidine being a 3-meU in the middle of the loop. The methyl group on the nitrogen 3 of the Uracil base (3-meU) would inhibit the ability of RNase A to cleave. At one end of the probe is a FAM fluorescent reporter and at the other a blackhole quencher (BHQ1), which are held in close proximity to one another by the stem structure. By demethylating the sole 3-meU on the probe’s loop, FTO would create a cleavage site for RNase A. Cleavage of the probe would destabilize the stem, separating the quencher from the fluorophore de-repressing the fluorescent signal. b. A methyl group on uridine inhibits RNase A. 100nM of the 3-me-uridine (black bars) or normal uridine (open bars) oligonucleotide is cleaved with increasing concentrations of RNase A in the reaction mix without FTO. Florescence emission is recorded for each substrate at room temperature every 30 minutes. Florescence signal is converted to a percentage of maximum cleavage, taking the normal uridine oligonucleotide in 1000 pg/μl of RNase A as the reference.

Figure 2. Validation of the assay looking at the demethylase activity of FTO.

Figure 2

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a. Florescence released from 100 nM of 3-me-uridine oligonucleotide is proportional to the amount of FTO. In the presence of 62.5 pg /μl of RNase A, initial velocities were determined by linear fit and were plotted against FTO concentration (nM). Reactions were performed in 50 mM Tris-HCl (pH7), 75 μM Fe(NH4)2(SO4)2, 300 μM 2-OG, 2 mM ascorbate and 50 μg/ml bovine serum albumin (BSA) at room temperature. Data fitted with an R2 = 0.986. b. Demonstration of FTO as a 2OG-dependent dioxygenase. Complete reaction contained the methylated oligonucleotide, FTO, RNase A, Fe(II), 2-OG and ascorbate. One of the components was removed from the reaction as indicated in the figure and florescent signals were monitored over time. In the enzyme negative controls, FTO was replaced with an equivalent amount of BSA. At the end of the experiment, florescent signal released from the complete reaction was taken as the reference for calculating the relative activity of FTO under various conditions. The left panel depicts the amount of fluorescence (RFU) generated by each reaction over time in minutes. The right panel depicts the calculated initial reaction velocity expressed as a percentage of maximal activity when all components are included. FTO activity is dependent upon the presence of 2-oxoglutarate (2-OG) and ascorbate, and is partially dependent on the presence of Fe(II).

Figure 3. The fluorescence assay can distinguish between wild-type and mutant R316Q FTO.

Figure 3

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Kinetic analyses of wild type FTO and mutant R316Q. Initial velocities of wild type (WT; filled circles) and mutant R316Q (open squares) FTO were determined by linear fits and plotted against increasing concentrations of FTO. The velocity of the reaction increases in proportion to WT FTO, while R316Q FTO has no discernible activity.

Figure 4. Determining the Km of FTO for 2-OG.

Figure 4

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Kinetic analysis of FTO in response to increasing concentrations of 2-OG. Initial velocities were determined by linear fit and plotted against 2-OG concentration. Michaelis-Menten plot was obtained and the Km of FTO for 2-OG was calculated to be 2.88 μM.

Results

Principal of the RNAase cleavage fluorescence assay

RNase A selectively cleaves the phosphodiester bond 3’ of unpaired pyrimidine residues [10]. The co-crystal structure of RNaseA and 3’ UMP suggested that a methyl group on the nitrogen 3 of the Uracil base (3-meU) would inhibit the ability of RNase A to cleave [11] and that this feature could be exploited to develop an assay for FTO activity. To this end, we have engineered a stem loop structure, where the stem is formed from DNA (to prevent cleavage by RNase A) and the loop from RNA, with the sole pyrimidine being a 3-meU in the middle of the loop. At one end of the probe is a FAM fluorescent reporter and at the other a blackhole quencher (BHQ1), which are held in close proximity to one another by the stem structure (Fig 1a). We and others have found that a stem-loop structure, as opposed to a linear RNA fragment, reduced background fluorescence, thereby increasing the signal to noise ratio [12–14]. Thus, we expected that by demethylating the sole 3-meU on the probe’s loop, FTO would create a cleavage site for RNase A. Cleavage of the probe would destabilize the stem, separating the quencher from the fluorophore de-repressing the fluorescent signal. In the presence of an excess of RNase A FTO activity would become the rate-limiting step in the development of the fluorescent signal. An otherwise identical unmethylated probe would test these assumptions as its fluorescence would be RNase A dependent but independent on FTO.

A 3 methyl Uracil is a poor substrate for RNase A

To test the prediction that a methyl group on third position of uracil would inhibit the ability of RNase A to cleave, we performed a dose response experiment comparing the ability of different concentrations of RNase A to cleave both the methylated and the unmethylated substrate. After initial optimization testing any interference on the florescence signal from components (supplementary Fig S1), in each reaction, 100 nM of either methylated or un-methylated substrate was mixed with 75 μM Fe(NH4)2(SO4)2, 300 μM 2-oxoglutarate, 2 mM ascorbate, 50 μg/ml bovine serum albumin (BSA) and increasing amounts of RNase A from 10-12 μg/μl to 0.1 μg/μl in 50 mM Tris buffer at pH7.0. Samples were prepared in duplicates and the FAM emission was measured for 30 min at a wavelength of 520 nm with excitation at 485 nm. At a concentration of 62.5pg/ul of RNAase A, there is selectivity for the unmethylated substrate over the methylated substrate, confirming the prediction (Fig 1b). We therefore selected a concentration of 62.5pg/ul RNase A for all of the subsequent experiments.

1uM FTO gives maximum activity

Next we performed a dose response experiment to identify the concentration of FTO that would give us the maximum response. As described above, 100 nM of the methylated substrate was incubated with the reaction mix with 62.5 pg/ul of RNase A, while FTO was introduced in increasing concentrations from 0 to 1000nM. FAM emission was measured for 30 min at a wavelength of 535 nm with excitation at 485 nm and the rate of the initial reaction was calculated by linear fit. We find that a concentration of 1uM FTO results in the maximum reaction rate (Fig 2a).

FTO demethylation reaction is 2-OG dependent and partially dependent on Fe (II)

In order to confirm that the fluorescence signal produce from the reaction is specific to FTO, we performed a number of controls. Using a systematic removal of individual components, we find that the production of a fluorescence signal is entirely dependent upon the presence of 2-OG, ascorbate and RNAase A, while removal Fe(II) reduces the produced signal (Fig 2b). Additionally, replacing FTO with an equivalent amount of BSA in the reaction does not produce any signal above background levels. RNase A by itself does produce a background fluorescence signal, increasing over time, indicating that RNAase A is still able to cleave after 3-methyl uracil, albeit at a far lower rate (Fig 2b).

The assay is able to distinguish between wildtype and mutant R316Q FTO

With all the components in place, we are able to perform the assay, with increasing concentrations of FTO increasing the rate of reaction. When we assay the activity of a known mutation of FTO, R316Q, which we have previously reported to be catalytically inactive [8], the rate of reaction in response to increasing concentrations of enzyme is equivalent to that of the BSA control (Fig 3). Thus our novel assay can distinguish between WT and a known FTO mutant.

Measuring the Km of 2-OG for FTO

Because 2-OG, a key intermediate in the citric-acid cycle, is a co-substrate of FTO, it is possible that FTO functions as a sensor for intracellular concentrations of this metabolite and thus cellular metabolism. We then use this assay examine the enzyme kinetics of FTO with regards to its usage of 2-OG. As described above, 100 nM of the methylated substrate was incubated with the reaction mix with 625 pg of RNase A per reaction and 1uM of FTO, while increasing concentrations of 2-OG from 0-600uM was introduced. FAM emission was measured for 30 min at a wavelength of 535 nm with excitation at 485 nm. FTO responds to 2-OG consistent with Michaelis–Menten kinetics, with the Km for 2-OG calculated to be 2.88uM (Fig 4).

Discussion

FTO is the first and most robust of the so-called ‘post-GWAS’ obesity genes, with SNPs in its first intron being associated with increased BMI and risk for obesity. It is important to note that as yet, no conclusive link has been made between the risk alleles and expression or function and FTO. Certainly, the weight of evidence from a multitude of animal models where FTO expression has been perturbed indicates some role for FTO in energy homeostasis [4, 15–18]. However, the physiological role of FTO and how this might link to the regulation of body-weight has yet to be determined. With the aim of understanding more about its biochemistry, we have successfully developed a fluorescence RNase cleavage assay for FTO.

Enzyme kinetics

A key aim in developing this assay was to ensure a rapid way of testing the function of any further human FTO mutations we might identify. We have done this previously at great cost and effort using a radioactive based assay [8, 9]. In this regard, the assay was certainly very successful, as we are clearly able to distinguish an R316Q FTO mutant from WT FTO. Crucially however, the sensitive and continuous nature of this fluorescence assay meant we were able to obtain some previously unknown kinetic information. Because FTO is implicated in obesity, is nutritionally regulated within the brain, and utilizes 2-OG, a key metabolite in the citric acid cycle, as a co-substrate, it was certainly plausible that FTO could act as a sensor for intracellular metabolism. Thus, we applied our assay to examine the enzyme kinetics of FTO with regards to its usage of 2-OG, arriving at a 2-OG KM value for FTO of 2.88uM. Since typical intracellular concentrations of 2-OG are measured to be more than 10-fold higher, around 50-100uM [19], it is unlikely that FTO’s physiological role is to sense 2-OG. The 2-OG KM value for FTO appears to be in the similar region as for other dioxygenases such as PHD2 (0.9uM) [20] and Hif1alpha (10uM) [21].

High throughput screening?

Another advantage of this fluorescence assay, is its ability to be performed on a microliter scale, thus making it amenable to a high throughput screen for enhancers or inhibitors. The question however, lies in whether FTO, given its ubiquitous expression, can even be considered a viable target? The severity of the phenotype seen in human and murine FTO deficiency clearly points to some fundamental role, particularly in early postnatal development [8, 17]. Yet, we have been able to use it to influence food intake by discretely manipulating its expression in certain regions of the brain [4]. We hypothesize that while FTO clearly has a broader biological function, it also has a role specifically within the hypothalamus to regulate food intake. So there is the age-old problem of selectivity and specificity.

A second question is whether one would screen for an enhancer or an inhibitor? Human genetic data indicate that while SNPs in intron 1 of FTO are unequivocally associated with obesity in multiple populations, it appears that loss of one functional copy of FTO in humans is compatible with being either lean or obese [9]. Mouse models of Fto deficiency, although far from being straightforward, have been a little more helpful in illuminating a role for FTO energy balance [4, 15–18]. Mice homozygous for a targeted deletion in Fto display a complex phenotype [17]. They are post-natally growth retarded with decreased fat and lean body mass, and although are born with a normal body-weight and at the expected Mendelian ratio, display 50% lethality by the time of weaning. Fto -/- mice appear to display hyperphagia and increased energy expenditure when corrected for lean body-mass [17].

Mice overexpressing Fto showed a dramatic increase in food intake resulting in a marked increase in body weight and fat mass when they were fed either chow or a high-fat diet (HFD) [16]. Although the increase in weight with the overexpression of Fto seems consistent with Fto deficiency resulting in a ‘lean’ phenotype, the increase in food intake seen in these mice is not [16]. Central nervous system (CNS) specific Fto deleted mice have now been generated [18]. Surprisingly, these brain-specific Fto deficient mice recapitulate the phenotype of the whole-body knock-outs, although this is yet to be exhaustively examined. This suggests that much of Fto’s function, including its link to the regulation of energy homeostasis (and in keeping with the observations by [4]), is mediated in the brain.

Physiological role?

As designed, this assay will only work to determine FTO’s ability to demethylate 3 me-U [3, 5], and not the recently described 6-methyl adenosine [6], as RNaseA is only able to cleave after pyrimidines. It also does not address the question about whether or not 3 me-U is its endogenous substrate in vivo, nor how this might play a role in energy homeostasis. This was never our intention when we undertook this project. It is however, useful to consider where 3meU naturally occurs. As it turns out, in as far as it has been measured, 3me-U is found primarily in ribosomal RNA [22]. 6me-A on the other hand is found primarily in mRNA [23].

Two other questions emerge regarding the utility of this assay; a) Would there be any difference in measured activity if endogenous FTO and not recombinant FTO were used in our assay? One would imagine that if any post-translational modifications were critical to FTO activity, the endogenous FTO would give an increase in activity. We are currently addressing this question. b) What are the prospects of using this assay to test the activity of FTO in biological samples? There are two reasons why this is unlikely to work for this specific assay. Firstly, there is the issue of getting the DNA/RNA hybrid reporter into the biological samples intact and secondly, once in the cell/tissue, there is the impossibility of protecting the reporter from the action of endogenous RNAases.

Conclusions

FTO, as we have discussed, influences the body-weight of a large proportion of the human population, yet its physiological role remains unknown. Understanding the biology of FTO and its downstream actions could potentially reveal novel therapeutic targets in our battle against the increasing epidemic of obesity. Our novel high-throughput assay will allow us to rapidly and economically screen for any potential functional interaction candidate binding partners may have, and will also allow us to revisit the library of naturally occurring human FTO mutations, with the potential to obtain further structure-function relations for FTO. Should it eventually prove appropriate, this assay would also lend itself to high throughput screening for enhancers or inhibitors of FTO function.

Supplementary Material

Figure S1

Acknowledgements

With thanks to R.Raines, Madison Wisconsin, USA for useful discussions. This study was supported by the UK Medical Research Council Centre for Obesity and Related metabolic Disorders (MRC-CORD) (GY, MM, SOR), the Wellcome Trust (HH, SOR, DR), the EU FP7-HEALTH-2009-241592 EurOCHIP (MM, SOR, GY) and EU FP7-HEALTH-266408 Full4Health (GY).

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Associated Data

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Supplementary Materials

Figure S1
EMS203458-supplement-Figure_S1.pdf (123.4KB, pdf)

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