Adipose METTL14‐Elicited N6‐Methyladenosine Promotes Obesity, Insulin Resistance, and NAFLD Through Suppressing β Adrenergic Signaling and Lipolysis

Abstract White adipose tissue (WAT) lipolysis releases free fatty acids as a key energy substance to support metabolism in fasting, cold exposure, and exercise. Atgl, in concert with Cgi‐58, catalyzes the first lipolytic reaction. The sympathetic nervous system (SNS) stimulates lipolysis via neurotransmitter norepinephrine that activates adipocyte β adrenergic receptors (Adrb1‐3). In obesity, adipose Adrb signaling and lipolysis are impaired, contributing to pathogenic WAT expansion; however, the underling mechanism remains poorly understood. Recent studies highlight importance of N6‐methyladenosine (m6A)‐based RNA modification in health and disease. METTL14 heterodimerizes with METTL3 to form an RNA methyltransferase complex that installs m6A in transcripts. Here, this work shows that adipose Mettl3 and Mettl14 are influenced by fasting, refeeding, and insulin, and are upregulated in high fat diet (HFD) induced obesity. Adipose Adrb2, Adrb3, Atgl, and Cgi‐58 transcript m6A contents are elevated in obesity. Mettl14 ablation decreases these transcripts’ m6A contents and increases their translations and protein levels in adipocytes, thereby increasing Adrb signaling and lipolysis. Mice with adipocyte‐specific deletion of Mettl14 are resistant to HFD‐induced obesity, insulin resistance, glucose intolerance, and nonalcoholic fatty liver disease (NAFLD). These results unravel a METTL14/m6A/translation pathway governing Adrb signaling and lipolysis. METTL14/m6A‐based epitranscriptomic reprogramming impairs adipose Adrb signaling and lipolysis, promoting obesity, NAFLD, and metabolic disease.


Figure S1. Mettl14
Δfat mice are normal on chow diet.A) C57BL/6J male mice (8 weeks old) were fasted overnight and then refed for 3 h.Mettl3 protein (normalized to Actb) and mRNA (normalized to 36B4) levels were measured in eWAT and iWAT by immunoblotting and qPCR, respectively (n=4 mice per group), as described in Figure 1A.a.u.: arbitrary unit.B) 3T3-L1 cells were differentiated into adipocytes and stimulated with 5 µg/ml insulin for the indicated durations.Cell extracts were immunoblotted with the indicated antibodies.C) C57BL/6J male mice (8 weeks old) were fed a HFD for 16 weeks and then fasted overnight and refed for 3 h.WAT extracts were immunoblotted with the indicated antibodies.D) Mettl14 mRNA levels were measured in iWAT and eWAT (24 weeks old) by qPCR and normalized to 36B4 levels.a.u.: arbitrary unit.Mettl14 f/f : n=5, Mettl14 Δfat : n=7.E) Primary adipocytes were isolated from eWAT, and total RNA was extracted for m6A dot blot assays.F) Tissues were harvested from Mettl14 f/f and Mettl14 Δfat male mice at 24 weeks of age.Tissue extracts were immunoblotted with antibodies to Mettl14 and Hsp90.G-H) Mettl14 f/f and Mettl14 Δfat male mice were placed on chow diet.G) Growth curves.Mettl14 f/f : n=7, Mettl14 Δfat : n=5.H) GTT and ITT at 18 weeks of age.Mettl14 f/f : n=7, Mettl14 Δfat : n=5.Data are presented as mean ± SEM. *p<0.05,**p<0.01,***p<0.001,Student's t test.Figure S5.Mettl14 cell-autonomously suppresses adipose lipolysis.A) WAT was isolated from male mice (8 weeks) and stimulated with isoproterenol (1 µM) for 3 h in the presence or absence of insulin (100 nM).Glycerol secretion rates were measured and normalized to WAT weights.B) MEFs were differentiated into adipocytes for 8 days using an adipose differentiation cocktail.Total TAG levels were measured and normalized to proteins (n=3 repeats per group).C) Comparation of mouse and human METTL14 amino acid sequences.D) 3T3-L1 cells were differentiated into adipocytes and transduced with AAV-CAG-METTL14 or AAV-CAG-GFP vectors (for 3 days).Cell lysates were immunoblotted with antibodies to METTL14 and Actb.E) MEFs were transduced with METTL14 or GFP lentiviral vectors and differentiated into adipocytes.Cell lysates were immunoblotted with antibodies to METTL14 and Actb.Data are presented as mean ± SEM.

Figure S7
. Adipose Mettl14 regulates the levels of lipolysis regulators and β-adrenergic signaling in adipocytes.A-B) Male mice (8 weeks) were fed a HFD for 4 weeks.iWAT and eWAT were isolated and stimulated with isoproterenol (1 µM) for 15 min.A) WAT extracts were immunoblotted with antibodies to phospho-HSL (pSer563, pSer660), and pSer563 and pSer660 levels were quantified and normalized to total Hsl levels (n=3 mice per group).a.u.: arbitrary unit.B) WAT extracts were immunoblotted with antibodies to pan-phospho-PKA substrates and Actb.C) MEF cells were differentiated into adipocytes for 8 days.mRNA levels were measured by qPCR and normalized to 36B4 levels (n=6 repeats per group).D) MEFs were differentiated into adipocytes in vitro for 6 days, serum-deprived for 8 h, and stimulated with isoproterenol (1 µM) for 15 min.Cell extracts were immunoblotted with the indicated antibodies.E) 3T3-L1 cells were differentiated into adipocytes and treated with STM2457 (5 µM) for 48 h.Cell extracts were immunoblotted with the indicated antibodies.Protein levels were quantified and normalized to Actb levels (n=3 repeats per group).Data are presented as mean ± SEM. *p<0.05,**p<0.01,Student's t test.

Figure S8
. Influences of Mettl14-mediated m6A modification on stability and translational efficiency of lipolysis-related transcripts.A) Primary adipocytes were isolated from eWAT (8 weeks old) to extract total RNA.RNA was immunoprecipitated with anti-m6A antibody (a copy of the results of Figure 6A) or IgG.Precipitated RNA was extracted and used to measure the indicated transcripts by qPCR (normalized to inputs, n=3 mice per group).B) MEF-derived adipocytes were transduced with AAV vectors, and cell extracts were immunoblotted with the indicated antibodies.C) SVF cells were isolated from iWAT (8 weeks old) and differentiated into adipocytes.Adipocytes were treated with actinomycin D (1 µM) for 0-6 h.Total RNA was extracted to measure mRNA abundance by qPCR (normalized to 18S levels).The results were presented as percentages of baseline values (n=3 repeats per group).a.u.: arbitrary unit.D) Schematic representation of OPP pulldown assays.Data are presented as mean ± SEM. *p<0.05,**p<0.01,ANOVA.

Figure S9
. Influences of Mettl14-mediated m6A modification on stability and translational efficiency of lipolysis-related transcripts.C57BL/6J male mice (8 weeks old) were fed a HFD for 10 weeks.A) eWAT extracts were immunoblotted with the indicated antibodies.B) eWAT mRNA levels were measured by qPCR (normalized to 36B4 levels, n=8 mice per group).C) iWAT and eWAT explants were isolated and stimulated with isoproterenol (1 µM, Iso) for 3 h.Glycerol-and FFA-releasing rates were measured and normalized to WAT weight.a.u.: arbitrary unit.Chow: n=7, HFD: n=6.D) 3T3-L1 cells were differentiated into adipocytes and stimulated with 5 µg/ml insulin for 16 h.Gene expression was measured by qPCR and normalized to 36B4 levels (n=4 repeats per group).E-F) Human visceral WAT samples were used to extract total RNA or prepare WAT extracts.E) Gene expression was measured by qPCR (normalized to 18S).Lean: n=9, obese: n=8.F) WAT extracts were immunoblotted with anti-METTL14 antibody.G) Obesogenic factors upregulate adipose METTL3/ and METTL14 that in turn install m6A in transcripts encoding β adrenergic (Adrb2/3) and lipolytic (Atgl/Cgi-58) pathway mediators.m6A methylation influences processing and suppresses translation of target mRNAs, thereby suppressing β adrenergic signaling (catecholamine resistance) and lipolysis to increase adipose expansion.Thus, adipose m6A-centric epitranscriptomic reprogramming promotes obesity, NAFLD, and metabolic disorders.

Figure S6 .
Figure S6.Adipose Mettl14 regulates the levels of lipolysis regulators and β-adrenergic signaling in adipocytes.Mettl14 f/f and Mettl14 Δfat males (8 weeks old) were fed a HFD for 16 weeks.A-B) eWAT was harvested for RNA-seq (n = 3).A) GO analyses of upregulated and downregulated genes.B) Gene