Regulatory role and mechanism of m⁶A RNA modification in human metabolic diseases

Abstract

Metabolic diseases caused by disorders in amino acids, glucose, lipid metabolism, and other metabolic risk factors show high incidences in young people, and current treatments are ineffective. N⁶-methyladenosine (m⁶A) RNA modification is a post-transcriptional regulation of gene expression with several effects on physiological processes and biological functions. Recent studies report that m⁶A RNA modification is involved in various metabolic pathways and development of common metabolic diseases, making it a potential disease-specific therapeutic target. This review explores components, mechanisms, and research methods of m⁶A RNA modification. In addition, we summarize the progress of research on m⁶A RNA modification in metabolism-related human diseases, including diabetes, obesity, non-alcoholic fatty liver disease, osteoporosis, and cancer. Furthermore, opportunities and the challenges facing basic research and clinical application of m⁶A RNA modification in metabolism-related human diseases are discussed. This review is meant to enhance our understanding of the molecular mechanisms, research methods, and clinical significance of m⁶A RNA modification in metabolism-related human diseases.

Graphical abstract

N⁶-methyladenosine (m⁶A) RNA modification plays critical roles in various metabolic pathways and development of common metabolic diseases. Zhang et al. summarize components, mechanisms, and research methods of m⁶A RNA modification, and they also review research progress and clinical significance and discuss future directions of m⁶A RNA modification in human metabolic diseases.

Introduction

More than 100 types of chemical modifications in cellular RNAs have been reported during the past 6 decades.¹ Several methylation modifications occur in eukaryotic messenger RNA (mRNA), including N⁷-methylguanosine, N⁶-methyl-2′-O-methyladenosine, 2′-O-methylation, N⁶-methyladenosine (m⁶A), and 5-methylcytosine.² Notably, m⁶A is the most common RNA modification type. It was first reported in mRNAs from eukaryotes in the early 1970s.³ m⁶A RNA modification mainly occurs in the RRACH sequence,^4,5 which is mainly found near stop codons, and in 3′ untranslated regions (UTRs) and long internal exons in mRNAs.^6,7 Advances in high-throughput sequencing technology and gradual progress of epigenetic research have enabled the study of m⁶A RNA modifications in a variety of non-coding RNAs (ncRNAs), including ribosomal RNA (rRNA), transfer RNA (tRNA), small nuclear RNA (snRNA), microRNA (miRNA), and long ncRNA (lncRNA).^8,9 Notably, m⁶A RNA modification is a dynamic and reversible event that is catalyzed by a collection of enzymes, including methyltransferase “writers,” demethylase “erasers,” and “readers” that recognize such modifications.^10,11 m⁶A modifications are implicated in most steps of target RNA metabolism, including RNA maturation, splicing, export and folding, translation, and stability of RNA, thus modulating the downstream signaling pathway and physiological function.^12,13

Metabolic diseases caused by disorders in amino acids, glucose, lipid metabolism, and other metabolic disorders, including obesity, type 2 diabetes (T2D), non-alcoholic fatty liver disease (NAFLD), hypertension, atherosclerosis, chronic kidney disease, cardiovascular disease, are a global health burden.¹⁴ Several studies report that cancer is a type of metabolic disease, which may shift metabolic pathways to facilitate uptake and incorporation of nutrients into cell building blocks, such as nucleotides, amino acids, and lipids required by highly proliferating cells.^15,16 Although various approaches have been developed to prevent and treat these metabolic diseases, they have limited efficacy. The potentially important role of the m⁶A RNA modification in the development and progression of human metabolic disease is an emerging field of study.¹⁷

In this review, we summarize the recent progress in the study on the role and molecular mechanisms of m⁶A RNA modification in diseases associated with metabolism.

Mechanisms of m⁶A RNA modification

Regulators of m⁶A RNA modification can be classified into three types: writers, erasers, and readers. These enzymatic proteins are implicated in installing, removing, or recognizing m⁶A on mRNAs or ncRNAs, respectively (Figure 1).

Figure 1.

Mechanisms of m⁶A RNA modification

m⁶A methylation is catalyzed by the writer enzyme complex, which includes METTL3, METTL5, METTL14, METTL16, WTAP, RBM15, VIRMA/KIAA1429, ZC3H13, and ZCCHC4. The m⁶A modification is removed by the demethylase action of FTO, ALKBH3, and ALKBH5. Reader proteins recognize m⁶A and affect multiple downstream reactions, and they mainly include members of the YTH domain family (YTHDF1, YTHDF2, YTHDF3, YTHDC1, and YTHDC2), the HNRNP family (HNRNPA2B1 and HNRNPC), the IGF2BP family (IGF2BP1, IGF2BP2, and IGF2BP3), the eukaryotic initiation factor eIF3, and the proline-rich coiled-coil protein PRRC2A.

Writers comprise m⁶A methyltransferases, which catalyze m⁶A modification through a multicomponent methyltransferase complex that co-regulates transfer of methyl groups from S-adenosylmethionine to adenine bases in RNA.¹⁸ The main components of this complex include methyltransferase-like 3 (METTL3), methyltransferase-like 5 (METTL5), methyltransferase-like 14 (METTL14), methyltransferase-like 16 (METTL16), WT1-associated protein (WTAP), RNA-binding motif protein 15 (RBM15), Vir-like m⁶A methyltransferase associated (VIRMA, also known as KIAA1429), zinc finger CCCH-type containing 13 (ZC3H13), and zinc finger CCHC-type containing 4 (ZCCHC4). METTL3 was the first protein to be identified as an “m⁶A writer.”^19,20 METTL14 structurally supports METTL3, and they form the core methyltransferase complex for m⁶A modification.²¹ WTAP stabilizes the core complex and facilitates m⁶A by recruiting the complex to nuclear speckles.²² RBM15 promotes binding of METTL3 and WTAP, thus guiding the two proteins to their target sites.^20,23 VIRMA mainly promotes mRNA methylation modifications near the 3′ UTR and stop codon regions.²⁴ Alternatively, ZC3H13 and CBLL1 control nuclear m⁶A methylation by combining with other cofactors such as WTAP.²⁵ Recent studies report that ZCCHC4 is a methyltransferase involved in modification of the 28S rRNA.^26,27 In addition, METTL16 is an independent mRNA methyltransferase, implicated in maintaining mRNA stability and regulation of splicing, and its binding sites do not overlap with those of METTL3/METTL14 methylation complexes.²⁸ In addition, METTL16 can function alone and catalyze m⁶A on U6 snRNA, precursor (pre-)mRNAs, and ncRNAs.^29,30 METTL5 is implicated in 18S rRNA m⁶A modification (Table 1).³¹

Table 1.

Role of regulators in m⁶A RNA modification

Regulator	Function	Reference
m6A writers
METTL3	catalyzes m6A modification	19
METTL14	helps METTL3 to recognize the substrate	21
WTAP	contributes to localization of the METTL3-METTL14 heterodimer to the nuclear speckle	22
RBM15	binds the m6A-methylation complex and recruits it to specific sites in RNA	20,23
VIRMA/KIAA1429	VIRMA recruits the catalytic core components METTL3/METTL14/WTAP to guide methylation of a particular region	24
ZC3H13	anchors WTAP to the mRNA-binding factor in the nucleus	25
ZCCHC4	methylates human 28S rRNA and also interacts with a subset of mRNAs	26,27
METTL16	catalyzes m6A modification U6 snRNA and pre-mRNAs and ncRNAs independently	28,29
METTL5	promotes 18S rRNA m6A modification with TRMT112	31
m6A erasers
FTO	catalyzes m6A demethylation	32
ALKBH5	reverses m6A modifications oxidatively	33
ALKBH3	removes m6A modification on tRNA	34
m6A readers
YTHDF1	increases mRNA translation efficiency	35
YTHDF2	promotes mRNA degradation	36
YTHDF3	enhances translation and degradation by interacting with YTHDF1 and YTHDF2	35
YTHDC1	contributes to RNA splicing and export	37
YTHDC2	enhances translation of target RNA and decreases abundance of target RNA	38
HNRNPA2B1	mediates mRNA splicing and primary microRNA processing	39
HNRNPC	affects abundance and alternative splicing of target mRNAs	40
HNRNPG	regulates transcriptome-wide alternative splicing	41
IGF2BPs	enhances mRNA stability and translation	42
eIF3	enhances mRNA translation	43
PRRC2A	stabilizes Olig2 mRNA	44

Erasers comprise m⁶A demethylases, which remove m⁶A methyl groups from RNA. Three m⁶A demethylases have been reported, including fat mass and obesity-associated protein (FTO) and ALKB homologs ALKBH5 and ALKBH3.^32,34,45 The demethylation process involves oxidation of m⁶A to form N⁶-hydroxymethyladenosine (hm⁶A), conversion of the hm⁶A to N⁶-formyladenosine (f⁶A), and finally conversion of f⁶A to adenosine. FTO was the first protein to be identified as an “m⁶A eraser,” and it catalyzes m⁶A demethylation.³² ALKBH5 was the second RNA demethylase to be reported, and it reverses m⁶A modifications.³³ In addition, FTO can mediate m⁶Am (N⁶,2′-O-dimethyladenosine) demethylation. However, ALKBH5 is an m⁶A-specific demethylase in mRNA.⁴⁶ Recently, Chen et al.⁴⁷ reported that ALKBH3 plays a role as a demethylase of m⁶A modifications, and that ALKBH3 preferentially modifies tRNA over mRNA or rRNA (Table 1).³⁴

m⁶A readers comprise the YTH domain-containing family (YTHDF1, YTHDF2, YTHDF3, YTHDC1, and YTHDC2),^37,38,48,49 the heterogeneous nuclear ribonucleoprotein (HNRNP) family (HNRNPA2B1, HNRNPC, and HNRNPG),39, 40, 41^,50 insulin-like growth factor 2 mRNA-binding proteins (IGF2BP1, IGF2BP2, and IGF2BP3),⁴² eukaryotic initiation factor eIF3,^43,51 and proline-rich coiled-coil protein PRRC2A.⁴⁴ YTHDF1 interacts with initiation factors, thus promoting RNA translation initiation in cytosol.⁵² YTHDF2 regulates RNA degradation by binding to m⁶A-modified mRNA.³⁶ YTHDF3 promotes translation by enhancing protein synthesis together with YTHDF1 and regulates mRNA decay mediated by YTHDF2.^35,53 Furthermore, YTHDF1 and YTHDF2 recognize m⁶A-modified circRNAs and modulate circRNA expression.^54,55 YTHDC1 can affect subcellular localization of circRNA in an m⁶A-dependent manner.⁵⁶ Furthermore, IGF2BP1–IGF2BP3 enhance stability and translation of the target mRNAs by recognizing m⁶A modifications.⁴² HNRNPA2B1 recognizes primary (pri-)miRNA m⁶A marks and interacts with DiGeorge syndrome critical region 8 (DGCR8), a critical component of the canonical microprocessor complex, to stimulate miRNA processing, whereas HNRNPC recognizes m⁶A-dependent splicing in mRNA secondary structures.³⁹ Wu et al.⁴⁴ reported a novel m⁶A reader, PRRC2A, which stabilizes Olig2 mRNA by binding to a consensus GGACU motif in the Olig2 coding sequence in an m⁶A-dependent manner (Table 1).

Methods for m⁶A RNA modification research

Levels of m⁶A in RNA are determined by two-dimensional thin layer chromatography, m⁶A dot blots, and high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS).^57,58 Transcriptome-wide distribution of m⁶A is profiled by methylated RNA immunoprecipitation followed by high-throughput sequencing (MeRIP-seq or m⁶A-seq).⁶ In this method, mRNA or ncRNA is fragmented into 100-nt-long oligonucleotides and immunoprecipitated with a specific antibody against m⁶A. The precipitated RNAs are then subjected to high-throughput sequencing. In addition, methods with higher resolution, such as photo-crosslinking-assisted m⁶A sequencing (PA-m⁶A-seq) and site-specific cleavage and radioactive labeling followed by ligation-assisted extraction and thin-layer chromatography (SCARLET), have been developed.⁵⁹ The m⁶A individual nucleotide resolution crosslinking immunoprecipitation (miCLIP) method can detect m⁶A at a precise position, and it is a major advance in m⁶A research.⁶⁰ Recent studies developed antibody-free methods for global m⁶A detection, including MAZTER-seq,⁶¹ DART-seq (deamination adjacent to RNA modification targets),⁶² and m⁶A-SEAL (an m⁶A selective chemical labeling method).⁶³

Advances in CRISPR-based genome engineering allow determination of the functional role of changing the m⁶A modification site in many organisms.⁶⁴ Furthermore, Zhou et al.⁶⁵ developed an online tool, named the sequence-based m⁶A modification site predictor, for prediction of m⁶A modification sites on RNA sequences of interests. These methods allow exploration of the function and mechanisms of m⁶A modification.

Role of m⁶A modification in glucose metabolism-related diseases

Glucose metabolism is a complex and important source of energy in organisms through anaerobic fermentation, aerobic oxidation, and the pentose phosphate pathway. T2D is a complex metabolic disease characterized by hyperglycemia and dyslipidemia.⁶⁶ Recent studies report that m⁶A modification plays a critical role in the pathogenesis of T2D. In patients with T2D, glucose levels affect dynamic regulation of m⁶A. High glucose levels can simultaneously decrease FTO mRNA expression and increase expression of METTL3, METTL14, and WTAP methyltransferases. Forkhead box O1 (FOXO1) and glucose-6-phosphatase catalytic subunit 1 (G6PC) are key regulators in glucose homeostasis.^67,68 Diacylglycerol O-acyltransferase 2 (DGAT2) is required for synthesis and storage of intracellular triglycerides, which play a central role in lipid accumulation.⁶⁹ Notably, a high expression level of FTO induces mRNA expression of FOXO1, G6PC, and DGAT2, resulting in abnormal glucose and lipid metabolism.^70,71 Recent studies report that m⁶A modification is associated with β cell survival and insulin secretion, which are important for regulation of glucose levels in T2D patients. METTL3 suppresses hepatic insulin sensitivity through modification of fatty acid synthase (FASN) mRNA by m⁶A and promotion of fatty acid metabolism.⁷² METTL3 is downregulated under inflammatory and oxidative stress conditions. Deletion of METTL3 induces islet β cell failure and hyperglycemia.⁷³ METTL14 is implicated in β cell survival, differentiation, and insulin secretion. Knockdown of METTL14 in mice increases β cell death, alters β cell differentiation, and decreases β cell mass and insulin secretion, resulting in glucose intolerance.⁷⁴ Furthermore, depletion of m⁶A in-EndoC-βH1 decreases insulin secretion by decreasing AKT phosphorylation and pancreatic and duodenal homeobox 1 (PDX1) protein levels, which have been explored using β cell-specific METTL14 knockout mice.⁷⁵ These studies provide a theoretical basis for development of m⁶A-based molecular therapies to promote β cell survival and function in patients with diabetes. In summary, m⁶A modification plays an important role in glucose metabolism, which is associated with several human diseases, and is a potential therapeutic target (Table 2).

Table 2.

Functions of m⁶A RNA modification in glucose metabolism

m6A regulators	Function	Disease	Reference
FTO	high expression of FTO induced mRNA expression of FOXO1, G6PC, and DGAT2, which are involved in abnormal glucose	type 2 diabetes	70
METTL3	suppresses hepatic insulin sensitivity through m6A modification of FASN (fatty acid synthetase) mRNA and promoting fatty acid metabolism	type 2 diabetes	72
METTL14	knockdown of METTL14 decreases β cell mass and insulin secretion, eventually resulting in glucose intolerance	type 2 diabetes	74
METTL14	knockdown of METTL14 in β cells decreases AKT phosphorylation and PDX1 protein levels, resulting in a decrease in insulin secretion	type 2 diabetes	75

Role of m⁶A modification in lipid metabolism-related diseases

Lipid metabolism is a complex physiological process that is associated with nutrient adjustment, hormone regulation, and homeostasis, and it involves multiple molecular factors and signaling pathways.⁷⁶ Abnormal lipid metabolism is implicated in several diseases,⁷¹ with recent studies reporting that m⁶A modification plays a role in this relationship.

Low METTL3 activity in cell cultures decreases m⁶A modification of the peroxisome proliferator-activated receptor (PPAR)α gene, thus increasing its mRNA expression and extending the life of transcripts, which ultimately reduces lipid accumulation. Analysis shows that YTHDF2 binds to PPARα mRNA, thus mediating its stability and regulates lipid metabolism.⁷⁷ The zinc finger protein 217 (Zfp217) binds to YTHDF2 to activate transcription of the m⁶A demethylase FTO, thus promoting its interaction with m⁶A sites on various mRNAs, and it ultimately promotes adipose differentiation. These activities were confirmed through decreased levels of oil red O staining and lower mRNA expression of key adipogenic genes encoding PPARγ, lipoprotein lipase, and adiponectin in mouse embryonic fibroblasts.⁷⁸ Translation of mitochondrial carrier homology 2 (MTCH2) is mediated by m⁶A modification through an YTHDF1-dependent pathway, and it plays a role in regulating adipogenesis in intramuscular preadipocytes.⁷⁹ For m⁶A erasers, FTO enhances expression of JAK2 and promotes phosphorylation of STAT3, thus enhancing transcription of C/EBPβ implicated in early stages of adipocyte differentiation. Alternatively, YTHDF2 accelerates decay of JAK2 mRNA and attenuates JAK2-STAT3-C/EBPβ signaling.⁸⁰ RUNX1 partner transcriptional co-repressor 1 (RUNX1T1) is a regulator of adipogenesis.⁸¹ FTO controls splicing of RUNX1T1 exons by regulating m⁶A levels of its transcripts, thus regulating adipogenesis.⁸² Angiopoietin-like 4 (ANGPTL4) plays a role in the regulation of triglyceride clearance from the blood serum and in lipid metabolism.⁸³ FTO binds to ANGPTL4 mRNA and promotes its translation, thus enhancing intracellular lipolysis in mouse adipocytes.⁸⁴ The roles of m⁶A modification in lipid metabolism are summarized in Table 3.

Table 3.

Functions of m⁶A RNA modification in lipid metabolism

Regulators	Function	Mechanism	Reference
METTL3	METTL3 decreases PPARα m6A abundance and increases PPARα mRNA half-life and expression, reducing lipid accumulation	mRNA stability	77
YTHDF2/FTO	promotes adipose differentiation	gene expression	78
YTHDF1	YTHDF1 promotes mitochondrial carrier homology 2 (MTCH2) translation to regulate adipogenesis	translation	79
FTO/YTHDF2	FTO enhances expression of JAK2 and YTHDF2 directly targets JAK2 and accelerates mRNA decay	gene expression, mRNA decay	80
FTO	FTO controls splicing of RUNX1T1 mRNA by regulating m6A levels, regulating adipogenesis	mRNA splicing	82
FTO	FTO binds to Angptl4 to encode an adipokine that stimulates intracellular lipolysis in adipocytes	translation	84
ime4Δ	ime4Δ (yeast m6A methyltransferase gene deletion) cells showed a significant decrease in expression of the key genes involved in peroxisomal β-oxidation in yeast	gene expression	85
YTHDF1	facilitates translation of Wnt signaling effectors TCF7L2 and TCF4, which are required for maintenance of intestinal stem cells (ISCs) during regeneration and tumorigenesis	translation	86

Obesity is a common contributor to metabolic syndrome. At the cellular level, obesity is characterized by an increase in both cell size (hypertrophy) and the number of fat cells (hyperplasia).^87,88 Several studies report that m⁶A modification is involved in the development of obesity. The m⁶A writers WTAP, METTL3, and METTL14 promote cell cycle transition in mitotic clonal expansion; however, these methyltransferases are negatively correlated with adipogenesis.^89,90 Risk alleles in the m⁶A eraser FTO are common among people with high body mass index, and some of these single nucleotide polymorphisms are positively correlated with obesity.91, 92, 93, 94, 95 In addition, FTO promotes adipogenesis by repressing the Wnt/β-catenin signaling pathway in porcine intramuscular preadipocytes.⁹⁶

However, several studies report that m⁶A writers, erasers, and readers do not work alone. For example, FTO increases expression of autophagy-related 5 (ATG5) and autophagy-related 7 (ATG7) expression to repress formation of autophagosomes, thus inhibiting autophagy and adipogenesis. Alternatively, YTHDF2 decreases expression of ATG5 and ATG7 by shortening the half-life of their m⁶A-modified mRNAs.⁹⁷ Moreover, FTO regulates adipogenesis by controlling cell cycle progression in an m⁶A-YTHDF2-dependent manner. FTO knockdown significantly decreases expression of cell cycle regulators such as cyclin A2 (CCNA2) and cyclin-dependent kinase 2 (CDK2). YTHDF2 recognizes and destabilizes these mRNAs, leading to reduced protein expression, prolonged cell cycle progression, and suppressed adipogenesis.⁹⁸ Induced expression of YTHDF2 reverses demethylation of CCNA2 and CDK2 mRNAs induced by FTO.⁹⁹ Low levels of ZFP217, which is implicated in adipogenesis, increase expression of METTL3. Furthermore, knockdown of METTL3 rescues mitotic clonal expansion inhibited by ZFP217 small interfering RNA and promotes cyclin D1 (CCND1) expression. Moreover, YTHDF2 recognizes and degrades m⁶A-methylated CCND1 mRNA, resulting in downregulation of CCND1 and inhibition of adipogenesis.¹⁰⁰

Currently, prevalence of NAFLD is at epidemic proportions and is a common cause of chronic liver disease worldwide.¹⁰¹ NAFLD is characterized by hepatic steatosis, ballooning degeneration, and fatty retention of liver parenchyma cells, with no history of excessive alcohol intake.¹⁰² Hepatic steatosis, the unique pathological feature of NAFLD, is caused by metabolic dysregulation of de novo lipogenesis, fatty acid uptake and oxidation, and triglyceride transport.^103,104 Previous studies report that development of NAFLD is highly correlated with m⁶A alteration.¹⁰⁵ m⁶A modification is associated with fat accumulation both in vivo and in vitro. The m⁶A eraser FTO decreases mitochondrial content and increases triglyceride deposition by downregulating overall m⁶A levels. FTO activity is essential for fat metabolism in hepatocytes, indicating the importance of RNA modification in fat deposition.¹⁰⁶ FTO is implicated in hepatic oxidative stress and lipid deposition, which participate in the development of NAFLD.¹⁰⁷ Curcumin, the active ingredient in dietary turmeric, affects mRNA expression of the m⁶A writers METTL3 and METTL14 in weaned piglets. Analysis showed that increased m⁶A methylation levels alleviate lipopolysaccharide-induced liver injury and reverses disruption of lipid metabolism in the liver.¹⁰⁸ In another study, knockdown of METTL3 or YTHDF2 increases expression of PPARα mRNA and reduces lipid accumulation.⁷⁷ In summary, m⁶A modulators are potential therapeutic targets for NAFLD.

Osteoporosis is a common bone metabolic disease in older populations.¹⁰⁹ Recent studies report that m⁶A modification is involved in development of osteoporosis.^110,111 Conditional knockout of the m⁶A writer METTL3 in bone marrow mesenchymal stem cells in mice decreases bone mass with low osteogenic potential, and it increases marrow adiposity associated with enhanced adipogenic potential by reducing m⁶A methylation levels.¹¹¹ METTL3 knockdown in porcine bone marrow stem cells promotes adipogenesis and adipogenic differentiation by targeting the JAK1/STAT5/C/EBPβ pathway in an m⁶A-YTHDF2-dependent manner.¹¹² Upregulation of the m⁶A eraser FTO by growth differentiation factor 11 promotes differentiation of bone marrow mesenchymal stem cells into adipocytes and osteoblasts through demethylation of PPARγ (Table 4).¹¹⁰

Table 4.

Functions of m⁶A RNA modification in lipid metabolism-related human diseases

m6A regulators	Function	Disease	Reference
WTAP	WTAP, METTL3, and METTL14 are negatively related to adipogenesis	obesity	89
METTL3
METTL14
FTO	FTO alleles and some SNPs are positively associated with obesity	obesity	91,93, 94, 95
FTO	promotes adipogenesis by repressing the Wnt/β-catenin signaling pathway in porcine intramuscular preadipocytes	obesity	96
FTO	increases the autophagy-related protein expression of ATG5/ATG7 and weaken the formation of autophagosomes, thereby inhibiting autophagy and adipogenesis	obesity	97
FTO/YTHDF2	FTO knockdown markedly decreases the expression of the cell cycle regulators CCNA2 and CDK2; YTHDF2 recognizes and degrades these mRNAs and reduces their protein expression to suppress adipogenesis	obesity	98
METTL3	downregulates CCND1 and inhibits adipogenesis	obesity	100
FTO	downregulates overall m6A levels and decreases mitochondrial content and triglyceride deposition	NAFLD	106
FTO	associates with hepatic oxidative stress and lipid deposition	NAFLD	107
METTL3/METTL14	Increases m6A methylation level, thus improving lipopolysaccharide-induced liver injury and hepatic lipid metabolism disruption in the liver of piglets	NAFLD	108
METTL3/YTHDF2	knockdown of METTL3 or YTHDF2 increases the expression of PPARα mRNA, thereby decreasing lipid accumulation	NAFLD	77
METTL3/YTHDF2	deletion of METTL3 promotes adipogenesis and adipogenic differentiation by targeting the JAK1/STAT5/C/EBPβ pathway via an m6A-YTHDF2-dependent manner	osteoporosis	112
FTO	promotes the differentiation of adipocyte and osteoblasts from bone marrow mesenchymal stem cells via GDF11	osteoporosis	110
METTL3	increases the m6A methylated level of fatty acid synthase (Fasn), thereby promoting fatty acid metabolism and enhancing hepatic insulin sensitivity	type 2 diabetes	72

Dysregulated fatty acid metabolism is associated with insulin resistance in diabetes.¹¹³ Recent studies report that m⁶A modification is involved in fatty acid metabolism. For instance, Xie et al.⁷² reported that the m⁶A writer METTL3 increases m⁶A methylation level of FASN, which promotes fatty acid metabolism and enhances hepatic insulin sensitivity. Their study provides key information on blood glucose homeostasis and provides information on potential therapeutic targets for T2D patients. m⁶A modification of mRNA is well characterized in yeast cells, where peroxisomes are the only sites for fatty acid β-oxidation. A deletion strain of the yeast m⁶A methyltransferase ime4 showed significant decrease in expression of key genes involved in peroxisomal β-oxidation compared with wild-type yeast. This study provides a basis for exploring the role of m⁶A methylation in peroxisomal biology (Table 3).⁸⁵

Role of m⁶A modification in cancer metabolic reprograming

Reprogramming metabolism is an important feature of cancer pathogenesis.¹¹⁴ Cancer cells activate or inhibit metabolic pathways such as aerobic glycolysis (also known as the Warburg effect),¹¹⁵ disordered lipid metabolism,¹¹⁶ and glutamine-dependent anaplerosis to accelerate cell proliferation.¹¹⁷ Recent studies report that m⁶A modification plays an important role in regulation of metabolic reprogramming of cancer.

m⁶A modification modulates glycolysis in cancer cells

The Warburg effect is a key metabolic hallmark of cancer cells that is characterized by elevated activation of glycolysis followed by increased lactate fermentation.¹¹⁸ In colorectal cancer (CRC), the m⁶A methyltransferase METTL3 interacts with the 3′ UTR regions of hexokinase 2 (HK2) and glucose transporter GLUT1 mRNA and enhances their stability, thus activating the glycolysis pathway.¹¹⁹ Moreover, WTAP enhances stability of HK2 mRNA by binding with the 3′ UTR m⁶A site in gastric cancer.¹²⁰ Casein kinase 2 (CK2) is associated with glycolysis, and CK2α is an essential catalytic subunit of the holoenzyme. In bladder cancer, ALKBH5 specifically recognizes the m⁶A sites of the 3′ UTR in CK2α mRNA and reduces its stability, thus inhibiting cell glycolysis and proliferation.¹²¹ Furthermore, FTO triggers m⁶A demethylation of PKM2 mRNA and accelerates its translation, thus promoting hepatocellular carcinoma tumorigenesis.¹²² In addition, YTHDF1 facilitates translation of the Wnt signaling effectors TCF7L2 and TCF4, which are required for maintenance of intestinal stem cells during regeneration and tumorigenesis.⁸⁶ YTHDF2 weakens EGFR mRNA stability in an m⁶A-dependent manner and inhibits the MEK/ERK pathway, consequently impeding cell proliferation.¹²³ METTL3 enhances translation of IKBKB and RELA and activates the nuclear factor κB (NF-κB) pathway, thus promoting bladder cancer progression.¹²⁴ METTL14 overexpression inactivates the phosphatidylinositol 3-kinase (PI3K)/Akt pathway in high glucose-treated HK2 cells through PTEN-affected, HDAC5-mediated epithelial-to-mesenchymal transition (EMT) of renal tubular cells in diabetic kidney disease.¹²⁵ YTHDF2 binds directly to the m⁶A modification site in the 3′ UTR of 6-phosphogluconate dehydrogenase (6PGD) gene, thus promoting 6PGD mRNA translation in lung cancer cells.¹²⁶ Moreover, Li et al.¹²⁷ reported that m⁶A modification of the 5′ UTR region of PDK4 upregulates its translation, elongation, and mRNA stability by binding with the YTHDF1/eEF-2 complex and IGF2BP3, thus enhancing tumor growth and progression of cervical and liver cancers (Table 5). These studies indicate that m⁶A modification plays an important role in glucose metabolism by modulating glycolytic enzymes or associated signaling pathways.

Table 5.

Functions of m⁶A RNA modification in metabolic reprogramming of cancer

m6A regulators	Molecules	Function	Reference
Glucose metabolism
METTL3	HK2, GLUT1	METTL3 interacts with the 3′ UTR regions of HK2 and GLUT1 mRNA and enhances its stability, thereby activating the glycolysis pathway	119
ALKBH5	CK2α	ALKBH5 specifically recognizes the m6A sites of the 3′ UTR in CK2α mRNA and reduces its stability to inhibit cell glycolysis and proliferation	121
FTO	PKM2	FTO triggers the m6A demethylation of PKM2 mRNA and accelerates its translation, thus promoting hepatocellular carcinoma tumorigenesis	122
YTHDF1	Wnt signaling	YTHDF1 facilitates the translation of the Wnt signaling effectors TCF7L2 and TCF4, which are required for the maintenance of intestinal stem cells during regeneration and tumorigenesis	86
YTHDF2	MEK/ERK	YTHDF2 weakens EGFR mRNA stability in an m6A-dependent manner and thus impairs the MEK/ERK pathway and consequently impedes cell proliferation and growth	123
METTL3	NF-κB pathway	METTL3 enhances the translation of IKBKB and RELA and activates NF-κB pathway to promote bladder cancer progression	124
METTL14	PI3K/Akt	METTL14 overexpression inactivates the PI3K/Akt pathway in high glucose-treated HK2 cells via PTEN-regulated HDAC5-mediated EMT of renal tubular cells in diabetic kidney disease	125
YTHDF2	6PGD	YTHDF2 binds directly to the m6A modification site in the 3′ UTR of the 6-phosphogluconate dehydrogenase (6PGD) gene to promote 6PGD mRNA translation in lung cancer cells	126
Lipid metabolism
METTL3	miR-3619-5p/HDGF	METTL3 promotes the stability of lncRNA LINC00958 and activates the miR-3619-5p/HDGF axis to facilitate lipogenesis in HCC	128
FTO	SREBP1c/CIDEC	FTO increases lipid accumulation by activating the SREBP1c/CIDEC signaling pathway in an m6A-dependent manner in HepG2 cells	129
Glutamine metabolism
FTO, ALKBH5	D2-HG	FTO and ALKBH5 are α-KG-dependent dioxygenases that are competitively inhibited by structurally related metabolite d-2-hydorxyglutarate (D2-HG), leading to abnormal expression of isocitrate dehydrogenase 1 or 2 (IDH1/2)-mutant tumors	130,131
YTHDF1	GLS1	YTHDF1 accelerates GLS1 translation, a key enzyme of glutamine metabolism, and promotes colon cancer development	132

m⁶A modification affects lipid metabolism in cancer cells

Cancer cells change lipid metabolism to meet the malignant development demands, including synthesis of macromolecules, the main lipids for biogenesis of membranes and various signaling factors.¹³³ METTL3 promotes stability of lncRNA LINC00958 and activates the miR-3619-5p/HDGF axis, thus inducing lipogenesis in HCC. In addition, LINC00958 affects the expression of sterol regulatory element binding transcription factor 1 (SREBP1), FASN, stearoyl-coenzyme A (CoA) desaturase (SCD1), and acetyl-CoA carboxylase 1 (ACC1), which are implicated in lipogenesis.¹²⁸ FTO increases lipid accumulation by activating the SREBP1c/CIDEC signaling pathway in an m⁶A-dependent manner in liver hepatocellular carcinoma HepG2 cells (Table 5).¹²⁹ These findings show that m⁶A RNA modification is involved in the regulation of lipogenesis in cancer cells.

m⁶A modification affects amino acid metabolism in cancer cells

Dysregulation of metabolism of amino acids, including glutamine, serine, and glycine, which play a role as metabolic regulators, is implicated in cancer cell growth.^134,135 Glutamine, the most abundant free amino acid, participates in several of pathways in energy generation, macromolecular synthesis, and signal transmission in cancer cells by donating its nitrogen and carbon atoms.¹³⁶ Glutamine can be converted into α-KG to replenish the tricarboxylic acid (TCA) cycle through glutamate dehydrogenase (GLUD1) or transaminases.¹³⁷ FTO and ALKBH5 are α-KG-dependent dioxygenases and are competitively inhibited by the structurally related metabolite, d-2-hydorxyglutarate (D2-HG), leading to accumulation in isocitrate dehydrogenase 1 or 2 (IDH1/2) mutant tumors.^130,131 In addition, YTHDF1 accelerates glutaminase GLS1 translation, a key enzyme in glutamine metabolism, therefore promoting development of colon cancers (Table 5).¹³² These findings imply that m⁶A modification affects glutamine metabolism, resulting in cancer pathogenesis; however, further studies should explore the role of glutamine metabolism of development of different cancers. Moreover, mores studies should explore the role of m⁶A on other amino acid metabolism.

Conclusions and perspectives

Advances in RNA immunoprecipitation sequencing, high-throughput sequencing, and liquid chromatography have led to the identification of several novel RNA modifications, implicated in metabolic diseases and tumors. In this review, we explored the roles and mechanisms of m⁶A RNA modifications in the occurrence and development of diseases by regulating glucose, lipid, and amino acid metabolism. The m⁶A-mediated regulation of glucose, lipid, and amino acid metabolism is associated with metabolic diseases, including T2D, NAFLD, obesity, and cancer (Figure 2). The stability and translation of mRNA of key regulators involved in these metabolic pathways are regulated by m⁶A modification and various m⁶A readers, thus promoting metabolic disease progression. In addition, m⁶A modification of lncRNA participates in progression of metabolic diseases.¹³⁸ In turn, metabolites and metabolic pathways are involved in regulation of m⁶A RNA modification; for example, TCA cycle metabolites affect FTO activity, whereas iron and NADPH (the reduced form of nicotinamide adenine dinucleotide phosphate) affect ALKBH5 activity.¹³⁹

Figure 2.

Role of m⁶A regulators in human metabolic diseases

Obesity mainly involves dysregulation of lipid metabolism, in which the expression of METTL3, METTL14, and WTAP are downregulated, while FTO is upregulated. In T2D, the expression of METTL14 is downregulated and FTO is upregulated in glucose metabolism, with increased expression of METTL3 in fatty acid metabolism. Metabolic disruption in human cancers mainly involves glycolysis and RNA metabolism. High METTL3 activates glycolysis to promote cancer development. The expression of FTO, METTL3, and YTHDF2 are high in the process of NAFLD. In osteoporosis, METTL3 expression is high, while FTO is low.

Incidence of metabolic diseases in young people are increasing exponentially. Current therapies for these diseases are ineffective, and therefore there is need to urgently explore and develop disease-specific therapeutic targets.^17,140 FTO is an attractive target for cancer treatment.¹⁴¹ Rhein, radicicol, epigallocatechin gallate, entacapone, and meclofenamic acid (MA) are a group of compounds that inhibit FTO regulation of m⁶A levels and affect fat formation.^99,142, 143, 144 MO-I-500 inhibits m⁶A demethylase activity of FTO and inhibits colony formation of a triple-negative inflammatory breast cancer cell line.¹⁴⁵ Meclofenamic acid, a nonsteroidal anti-inflammatory drug, selectively inhibits FTO and inhibits GBM cell growth and survival.¹⁴⁶ R-2HG, a major metabolic product of mutant IDH1/2, inhibits FTO activity, thus inhibiting leukemic cell growth/survival and leukemia progression.¹⁴⁷ A more potent FTO inhibitor, FB23-2, inhibits acute myeloid leukemia (AML) progression in xenotransplanted mice.¹⁴⁸ In addition, IOX3, an inhibitor of hypoxia-inducible factor prolyl hydroxylases, can be used to treat immune-related diseases caused by the m⁶A eraser ALKBH5.^45,149 The methylation inhibitors cycloleucine and S-adenosylhomocysteine can be used to reduce m⁶A levels,^106,150 whereas a methyl donor, betaine, increases m⁶A levels.¹⁵¹

Resistance to chemoradiotherapy is a major challenge in tumor therapy. m⁶A RNA modification regulators can be used as prediction markers for individualized cancer therapy and for providing clues for overcoming therapeutic resistance in cancer. In addition, silencing of METTL3 leads to an increase in the sensitivity of glioblastoma stem cells (GSCs) to gamma irradiation and pancreatic cancer cells to anticancer reagents.¹⁵² Recent studies report that m⁶A modification is related to development of the immune system. Silencing of METTL3 inhibits interleukin (IL)-7 signaling in CD4⁺ T cells. METTL3-mediated m⁶A modification enhances TLR4/NF-κB signaling-induced cytokine production and stimulates T cell activation.¹⁵³ YTHDF1 is associated with cross-presentation of tumor antigens, cross-priming of CD8⁺ T cells, and PD-L1 checkpoint inhibition in dendritic cells.¹⁵⁴ Knockdown of FTO in melanoma cells sensitizes tumor cells to interferon gamma (IFNγ) in vitro and promotes melanoma response to anti-PD-1 antibody in mice.¹⁵⁵ These findings imply that m⁶A regulators can be combined with anti-PD-1/PD-L1 inhibitors to improve anticancer immunotherapy. The relationship between m⁶A RNA modification and metabolism should be further explored. Therefore, use of m⁶A methylation-related inhibitors is a potential strategy for treatment of obesity and other complex metabolic diseases.

In addition, variations in gut microbiota are correlated with m⁶A modifications in the cecum and the liver, which affect metabolism, inflammation, and antimicrobial responses in mice.¹⁵⁶ Molecular interactions among human gut microbiota, m⁶A methylation, and metabolic diseases should be explored in the future.