A Mouse Model for the Metabolic Effects of the Human Fat Mass and Obesity Associated FTO Gene

Human FTO gene variants are associated with body mass index and type 2 diabetes. Because the obesity-associated SNPs are intronic, it is unclear whether changes in FTO expression or splicing are the cause of obesity or if regulatory elements within intron 1 influence upstream or downstream genes. We tested the idea that FTO itself is involved in obesity. We show that a dominant point mutation in the mouse Fto gene results in reduced fat mass, increased energy expenditure, and unchanged physical activity. Exposure to a high-fat diet enhances lean mass and lowers fat mass relative to control mice. Biochemical studies suggest the mutation occurs in a structurally novel domain and modifies FTO function, possibly by altering its dimerisation state. Gene expression profiling revealed increased expression of some fat and carbohydrate metabolism genes and an improved inflammatory profile in white adipose tissue of mutant mice. These data provide direct functional evidence that FTO is a causal gene underlying obesity. Compared to the reported mouse FTO knockout, our model more accurately reflects the effect of human FTO variants; we observe a heterozygous as well as homozygous phenotype, a smaller difference in weight and adiposity, and our mice do not show perinatal lethality or an age-related reduction in size and length. Our model suggests that a search for human coding mutations in FTO may be informative and that inhibition of FTO activity is a possible target for the treatment of morbid obesity.     

Pharmacological Inhibition of FTO

In 2007, a genome wide association study identified a SNP in intron one of the gene encoding human FTO that was associated with increased body mass index. Homozygous risk allele carriers are on average three kg heavier than those homozygous for the protective allele. FTO is a DNA/RNA demethylase, however, how this function affects body weight, if at all, is unknown. Here we aimed to pharmacologically inhibit FTO to examine the effect of its demethylase function in vitro and in vivo as a first step in evaluating the therapeutic potential of FTO. We showed that IOX3, a known inhibitor of the HIF prolyl hydroxylases, decreased protein expression of FTO (in C2C12 cells) and reduced maximal respiration rate in vitro. However, FTO protein levels were not significantly altered by treatment of mice with IOX3 at 60 mg/kg every two days. This treatment did not affect body weight, or RER, but did significantly reduce bone mineral density and content and alter adipose tissue distribution. Future compounds designed to selectively inhibit FTO’s demethylase activity could be therapeutically useful for the treatment of obesity.     

N6-methyladenosine demethylase FTO impairs hepatic ischemia–reperfusion injury via inhibiting Drp1-mediated mitochondrial fragmentation

Despite N6-methyladenosine (m6A) is functionally important in various biological processes, its role and the underlying regulatory mechanism in the liver remain largely unexplored. In the present study, we showed that fat mass and obesity-associated protein (FTO, an m6A demethylase) was involved in mitochondrial function during hepatic ischemia–reperfusion injury (HIRI). We found that the expression of m6A demethylase FTO was decreased during HIRI. In contrast, the level of m6A methylated RNA was enhanced. Adeno-associated virus-mediated liver-specific overexpression of FTO (AAV8-TBG-FTO) ameliorated the HIRI, repressed the elevated level of m6A methylated RNA, and alleviated liver oxidative stress and mitochondrial fragmentation in vivo and in vitro. Moreover, dynamin-related protein 1 (Drp1) was a downstream target of FTO in the progression of HIRI. FTO contributed to the hepatic protective effect via demethylating the mRNA of Drp1 and impairing the Drp1-mediated mitochondrial fragmentation. Collectively, our findings demonstrated the functional importance of FTO-dependent hepatic m6A methylation during HIRI and provided valuable insights into the therapeutic mechanisms of FTO.Subject terms: Mechanisms of disease, RNA modification     

Early hypothalamic FTO overexpression in response to maternal obesity--potential contribution to postweaning hyperphagia

Background

Intrauterine and postnatal overnutrition program hyperphagia, adiposity and glucose intolerance in offspring. Single-nucleotide polymorphisms (SNPs) of the fat mass and obesity associated (FTO) gene have been linked to increased risk of obesity. FTO is highly expressed in hypothalamic regions critical for energy balance and hyperphagic phenotypes were linked with FTO SNPs. As nutrition during fetal development can influence the expression of genes involved in metabolic function, we investigated the impact of maternal obesity on FTO.

Methods

Female Sprague Dawley rats were exposed to chow or high fat diet (HFD) for 5 weeks before mating, throughout gestation and lactation. On postnatal day 1 (PND1), some litters were adjusted to 3 pups (vs. 12 control) to induce postnatal overnutrition. At PND20, rats were weaned onto chow or HFD for 15 weeks. FTO mRNA expression in the hypothalamus and liver, as well as hepatic markers of lipid metabolism were measured.

Results

At weaning, hypothalamic FTO mRNA expression was increased significantly in offspring of obese mothers and FTO was correlated with both visceral and epididymal fat mass (P<0.05); body weight approached significance (P = 0.07). Hepatic FTO and Fatty Acid Synthase mRNA expression were decreased by maternal obesity. At 18 weeks, FTO mRNA expression did not differ between groups; however body weight was significantly correlated with hypothalamic FTO. Postnatal HFD feeding significantly reduced hepatic Carnitine Palmitoyltransferase-1a but did not affect the expression of other hepatic markers investigated. FTO was not affected by chronic HFD feeding.

Significance

Maternal obesity significantly impacted FTO expression in both hypothalamus and liver at weaning. Early overexpression of hypothalamic FTO correlated with increased adiposity and later food intake of siblings exposed to HFD suggesting upregulation of FTO may contribute to subsequent hyperphagia, in line with some human data. No effect of maternal obesity was observed on FTO in adulthood.     

Ubiquitination Regulates the Proteasomal Degradation and Nuclear Translocation of the Fat Mass and Obesity-Associated (FTO) Protein

Genetic polymorphisms in the fat mass and obesity-associated (FTO) gene have been strongly associated with obesity in humans. The cellular level of FTO is tightly regulated, with alterations in its expression influencing energy metabolism, food intake and body weight. Although the proteasome system is involved, the cellular mechanism underlying FTO protein turnover remains unknown. Here, we report that FTO undergoes post-translational ubiquitination on Lys-216. Knock-in HeLa cells harboring the ubiquitin-deficient K216R mutation displayed a slower rate of FTO turnover, resulting in an increase in the level of FTO as well as enhanced phosphorylation of the ribosomal S6 kinase. Surprisingly, we also found that K216R mutation reduced the level of nuclear FTO and completely abolished the nuclear translocation of FTO in response to amino acid starvation. Collectively, our results reveal the functional importance of ubiquitination in controlling FTO expression and localization, which may be crucial for determining body mass and composition.     

FTO demethylase activity is essential for normal bone growth and bone mineralization in mice

The Fto gene locus has been linked to increased body weight and obesity in human population studies, but the role of the actual FTO protein in adiposity has remained controversial. Complete loss of FTO protein in mouse and of FTO function in human patients has multiple and variable effects. To determine which effects are due to the ability of FTO to demethylate mRNA, we genetically engineered a mouse with a catalytically inactive form of FTO. Our results demonstrate that FTO catalytic activity is not required for normal body composition although it is required for normal body size and viability. Strikingly, it is also essential for normal bone growth and mineralization, a previously unreported FTO function.     

The expression of FTO in human adipose tissue is influenced by fat depot,adiposity, and insulin sensitivity

Objective: The fat mass and obesity associated (FTO) gene is related to obesity, but the regulation of FTO expression in adipose tissue is not fully understood. We investigated FTO expression in paired subcutaneous and omental adipose tissues (SAT and OAT) from healthy women undergoing gynecological surgeries, and its relation with adiposity and insulin sensitivity. Design and Methods: FTO expression in SAT of type 2 diabetic patients treated or not with Rosiglitazone was also compared. Results: Both the mRNA and protein levels of FTO were higher in OAT from women than in SAT. Only OAT FTO protein levels negatively correlated with BMI and body fat mass, whereas SAT FTO mRNA levels were negatively correlated with subcutaneous fat deposition. In addition, SAT FTO mRNA and protein levels were increased in insulin resistant women (high HOMA) compared to insulin sensitive women (low HOMA), whereas OAT FTO expression was not different between these two subgroups. Interestingly, FTO mRNA levels were increased in SAT of type 2 diabetic patients, and treatment of diabetics with Rosiglitazone improved insulin sensitivity and reduced SAT FTO mRNA levels. Lastly, FTO expression was transiently increased in the early phase of 3T3‐L1 cell differentiation, which coincides with the induction of PPARγ2 expression. However, partial reduction of FTO did not impact PPARγ2 expression and adipocyte differentiation. Conclusion: Therefore, FTO gene expression is higher in OAT than in SAT in lean to moderately obese women. OAT FTO expression is associated with adiposity, whereas SAT FTO expression is associated with insulin sensitivity. These associations are independent of an effect of FTO on adipocyte differentiation.     

Molecular regulatory mechanisms underlying the adaptability of polyploid plants

Polyploidization influences the genetic composition and gene expression of an organism. This multi-level genetic change allows the formation of new regulatory pathways leading to increased adaptability. Although both forms of polyploidization provide advantages, autopolyploids were long thought to have little impact on plant divergence compared to allopolyploids due to their formation through genome duplication only, rather than in combination with hybridization. Recent advances have begun to clarify the molecular regulatory mechanisms such as microRNAs, alternative splicing, RNA-binding proteins, histone modifications, chromatin remodelling, DNA methylation, and N⁶-methyladenosine (m6A) RNA methylation underlying the evolutionary success of polyploids. Such research is expanding our understanding of the evolutionary adaptability of polyploids and the regulatory pathways that allow adaptive plasticity in a variety of plant species. Herein we review the roles of individual molecular regulatory mechanisms and their potential synergistic pathways underlying plant evolution and adaptation. Notably, increasing interest in m6A methylation has provided a new component in potential mechanistic coordination that is still predominantly unexplored. Future research should attempt to identify and functionally characterize the evolutionary impact of both individual and synergistic pathways in polyploid plant species.     

The m6A mRNA demethylase FTO in granulosa cells retards FOS-dependent ovarian aging

Multifunctional N6-methyladenosine (m6A) has been revealed to be an important epigenetic component in various physiological and pathological processes, but its role in female ovarian aging remains unclear. Thus, we demonstrated m6A demethylase FTO downregulation and the ensuing increased m6A in granulosa cells (GCs) of human aged ovaries, while FTO-knockdown GCs showed faster aging-related phenotypes mediated. Using the m6A-RNA-sequence technique (m6A-seq), increased m6A was found in the FOS-mRNA-3′UTR, which is suggested to be an erasing target of FTO that slows the degradation of FOS-mRNA to upregulate FOS expression in GCs, eventually resulting in GC-mediated ovarian aging. FTO acts as a senescence-retarding protein via m6A, and FOS knockdown significantly alleviates the aging of FTO-knockdown GCs. Altogether, the abovementioned results indicate that FTO in GCs retards FOS-dependent ovarian aging, which is a potential diagnostic and therapeutic target against ovarian aging and age-related reproductive diseases.Subject terms: RNA modification, Infertility     

Methionine restriction restores a younger metabolic phenotype in adult mice with alterations in fibroblast growth factor 21

Methionine restriction (MR) decreases body weight and adiposity and improves glucose homeostasis in rodents. Similar to caloric restriction, MR extends lifespan, but is accompanied by increased food intake and energy expenditure. Most studies have examined MR in young animals; therefore, the aim of this study was to investigate the ability of MR to reverse age‐induced obesity and insulin resistance in adult animals. Male C57BL/6J mice aged 2 and 12 months old were fed MR (0.172% methionine) or control diet (0.86% methionine) for 8 weeks or 48 h. Food intake and whole‐body physiology were assessed and serum/tissues analyzed biochemically. Methionine restriction in 12‐month‐old mice completely reversed age‐induced alterations in body weight, adiposity, physical activity, and glucose tolerance to the levels measured in healthy 2‐month‐old control‐fed mice. This was despite a significant increase in food intake in 12‐month‐old MR‐fed mice. Methionine restriction decreased hepatic lipogenic gene expression and caused a remodeling of lipid metabolism in white adipose tissue, alongside increased insulin‐induced phosphorylation of the insulin receptor (IR) and Akt in peripheral tissues. Mice restricted of methionine exhibited increased circulating and hepatic gene expression levels of FGF21, phosphorylation of eIF2a, and expression of ATF4, with a concomitant decrease in IRE1α phosphorylation. Short‐term 48‐h MR treatment increased hepatic FGF21 expression/secretion and insulin signaling and improved whole‐body glucose homeostasis without affecting body weight. Our findings suggest that MR feeding can reverse the negative effects of aging on body mass, adiposity, and insulin resistance through an FGF21 mechanism. These findings implicate MR dietary intervention as a viable therapy for age‐induced metabolic syndrome in adult humans.     

The fat mass and obesity associated gene FTO functions in the brain to regulate postnatal growth in mice

FTO (fat mass and obesity associated) was identified as an obesity-susceptibility gene by several independent large-scale genome association studies. A cluster of SNPs (single nucleotide polymorphism) located in the first intron of FTO was found to be significantly associated with obesity-related traits, such as body mass index, hip circumference, and body weight. FTO encodes a protein with a novel C-terminal α-helical domain and an N-terminal double-strand β-helix domain which is conserved in Fe(II) and 2-oxoglutarate-dependent oxygenase family. In vitro, FTO protein can demethylate single-stranded DNA or RNA with a preference for 3-methylthymine or 3-methyluracil. Its physiological substrates and function, however, remain to be defined. Here we report the generation and analysis of mice carrying a conditional deletion allele of Fto. Our results demonstrate that Fto plays an essential role in postnatal growth. The mice lacking Fto completely display immediate postnatal growth retardation with shorter body length, lower body weight, and lower bone mineral density than control mice, but their body compositions are relatively normal. Consistent with the growth retardation, the Fto mutant mice have reduced serum levels of IGF-1. Moreover, despite the ubiquitous expression of Fto, its specific deletion in the nervous system results in similar phenotypes as the whole body deletion, indicating that Fto functions in the central nerve system to regulate postnatal growth.     

The role of RNA adenosine demethylases in the control of gene expression

RNA modifications are being recognized as an essential factor in gene expression regulation. They play essential roles in germ line development, differentiation and disease. In eukaryotic mRNAs, N⁶-adenosine methylation (m⁶A) is the most prevalent internal chemical modification identified to date. The m⁶A pathway involves factors called writers, readers and erasers. m⁶A thus offers an interesting concept of dynamic reversible modification with implications in fine-tuning the cellular metabolism. In mammals, FTO and ALKBH5 have been initially identified as m⁶A erasers. Recently, FTO m⁶A specificity has been debated as new reports identify FTO targeting N⁶,2′-O-dimethyladenosine (m⁶A_m). The two adenosine demethylases have diverse roles in the metabolism of mRNAs and their activity is involved in key processes, such as embryogenesis, disease or infection. In this article, we review the current knowledge of their function and mechanisms and discuss the existing contradictions in the field. This article is part of a Special Issue entitled: mRNA modifications in gene expression control edited by Dr. Soller Matthias and Dr. Fray Rupert.     

Regulation of Fto/Ftm gene expression in mice and humans

Two recent, large whole-genome association studies (GWAS) in European populations have associated a approximately 47-kb region that contains part of the FTO gene with high body mass index (BMI). The functions of FTO and adjacent FTM in human biology are not clear. We examined expression of these genes in organs of mice segregating for monogenic obesity mutations, exposed to underfeeding/overfeeding, and to 4 degrees C. Fto/Ftm expression was reduced in mesenteric adipose tissue of mice segregating for the Ay, Lep ob, Lepr db, Cpe fat, or tub mutations, and there was a similar trend in other tissues. These effects were not due to adiposity per se. Hypothalamic Fto and Ftm expression were decreased by fasting in lean and obese animals and by cold exposure in lean mice. The fact that responses of Fto and Ftm expression to these manipulations were almost indistinguishable suggested that the genes might be coregulated. The putative overlapping regulatory region contains at least two canonical CUTL1 binding sites. One of these nominal CUTL1 sites includes rs8050136, a SNP associated with high body mass. The A allele of rs8050136 preferentially bound CUTL1[corrected] in human fibroblast DNA. 70% knockdown of CUTL1 expression in human fibroblasts decreased FTO and FTM expression by 90 and 65%, respectively. Animals and humans with various genetic interruptions of FTO or FTM have phenotypes reminiscent of aspects of the Bardet-Biedl obesity syndrome, a confirmed "ciliopathy." FTM has recently been shown to be a ciliary basal body protein.     

Arginase Inhibition Ameliorates Hepatic Metabolic Abnormalities in Obese Mice

Objectives

We examined whether arginase inhibition influences hepatic metabolic pathways and whole body adiposity in diet-induced obesity.

Methods and Results

After obesity induction by a high fat diet (HFD), mice were fed either the HFD or the HFD with an arginase inhibitor, N^ω-hydroxy-nor-L-arginine (nor-NOHA). Nor-NOHA significantly prevented HFD-induced increases in body, liver, and visceral fat tissue weight, and ameliorated abnormal lipid profiles. Furthermore, nor-NOHA treatment reduced lipid accumulation in oleic acid-induced hepatic steatosis in vitro. Arginase inhibition increased hepatic nitric oxide (NO) in HFD-fed mice and HepG2 cells, and reversed the elevated mRNA expression of hepatic genes in lipid metabolism. Expression of phosphorylated 5′ AMPK-activated protein kinase α was increased by arginase inhibition in the mouse livers and HepG2 cells.

Conclusions

Arginase inhibition ameliorated obesity-induced hepatic lipid abnormalities and whole body adiposity, possibly as a result of increased hepatic NO production and subsequent activation of metabolic pathways involved in hepatic triglyceride metabolism and mitochondrial function.     

m^6 A RNA甲基化修饰异常在肿瘤中的作用

N6-甲基腺嘌呤(N6-methyladenosine,m6A)是真核生物信使RNA(messenger RNA,mRNA)含量最多的化学修饰之一。m6A修饰主要由m6A甲基转移酶(methyltransferase)催化,m6A去甲基酶(demethylase)去除,并由m6A结合蛋白(binding protein)识别。它广泛参与调控mRNA剪接、加工、翻译和降解等生命周期的各个阶段,且与肥胖和肿瘤等多种疾病及异常的生理功能相关。近年的研究发现,肿瘤中m6A相关蛋白质(METTL3/14、WTAP、FTO、ALKBH5、YTHDFs)的异常表达,引发m6A甲基化的失调,调控致癌基因和抑癌基因的表达参与肿瘤的发生与发展,并与患者预后不良密切相关。随着RNA免疫沉淀测序技术与高通量测序技术和液相色谱等检测技术的快速发展,有关m6A在肿瘤发生发展中的作用机制研究的进展迅猛,靶向m6A也成为肿瘤临床治疗的新方向。本文重点对m6A RNA甲基化相关因子在癌症发生发展中的作用及机制进行综述,总结m6A RNA甲基化检测技术的最新进展,梳理现有文献报道的脱甲基酶抑制剂大黄酸、甲氯芬那酸2(meclofenamic acid2,MA2)和右旋羟戊二酸(R-2-hydroxyglutarate,R-2HG)等在肿瘤靶向治疗中的运用,为以m6A RNA甲基化为切入点的肿瘤防治研究提供思路与理论参考。