FTO Regulated Intramuscular Fat by Targeting APMAP Gene via an m6A-YTHDF2-dependent Manner in Rex Rabbits

N6-methyladenosine (m6A) regulates fat development in many ways. Low intramuscular fat (IMF) in rabbit meat seriously affects consumption. In order to improve meat quality, we explored the law of IMF deposition. FTO could increase the expression of APMAP and adipocyte differentiation through methylation. However, interference YTHDF2 can partially recover the influence of interference FTO on the APMAP gene and adipocyte differentiation. APMAP promoted the differentiation of adipocytes. Analysis of IMF and APMAP expression showed IMF content is positive with the expression level of the APMAP gene (p < 0.01). Conclusion: Together, FTO can regulate intramuscular fat by targeting the APMAP gene via an m6A-YTHDF2-dependent manner in Rex rabbits. The result provides a theoretical basis for the molecular breeding of rabbits.


Introduction
Meat contains most minerals, vitamins, essential fatty acids, and other nutrients that human beings need [1]. The flavor of meat food is the main factor affecting consumers' choice and fatty acids in intramuscular fat can affect the flavor of meat food [2,3]. The content and composition of IMF is a very important symbol of meat quality and IMF influences the flavor, juiciness, and tenderness of meat [4]. Adipocyte differentiation and proliferation form fat deposition. Therefore, understanding the mechanism of adipogenesis could provide a scientific basis for breeding rabbits with high intramuscular fat.
M 6 A was discovered in the 1970s [5,6] and involved almost all aspects of RNA metabolism. Demethylases (FTO) [7,8], methylase [5], and methylation recognition enzyme (YTHDF2) [9][10][11][12] are the main factors causing m 6 A modification. The FTO gene is the first gene [13,14] that regulates fat deposition [15,16]. Studies indicated knockout of FTO inhibited fat deposition in mouse liver [17] and overexpression of FTO promoted adipogenesis in cells [18]. YTHDF2 has been found to regulate the translation of m 6 A containing mRNA [19]. However, it is not clear whether FTO and YTHDF2 regulate adipogenesis through m 6 A modification. So, it is important to explore the regulatory pathway of FTO on fat deposition.
APMAP contains 415 amino acids [20]. Studies found APMAP participated in the material exchange between the environment and mature adipocytes and regulates adipogenesis [21]. In addition, interference APMAP reduced fat deposition [21,22], which indicated APMAP was a crucial factor in adipogenesis. However, the role of APMAP in adipogenesis has not been reported yet.
In this study, we found that the expression level of the APMAP gene was higher in the muscle tissue of rabbits with high fat content. Further study showed FTO knockdown decreased APMAP expression by identification function of YTHDF2. In addition, we found that APMAP promoted adipocyte differentiation. Finally, the results of tissue PCR and intramuscular fat content showed that they were positively correlated (r = 0.844, p < 0.01). Our study provided a new way to breed Rex rabbits with high-quality meat.

Animals
The adipose tissues were isolated from newborn rabbits' perirenal fat after rabbits were slaughtered humanely. Adipose tissues were cut in PBS and digested in a 37°C water bath for 1 h. Finally, preadipocytes were obtained after filtering and centrifuging the mixture. Longissimus lumborum and perirenal fat were rapidly separated from 18 female Rex rabbits who were aged 35 days, 75 days, and 165 days. After separating the sample, we quickly put it in liquid nitrogen for 15 min and kept it in the −80°C refrigerator for a long time to extract mRNA. In addition, another 15 g longissimus lumborum was isolated to keep in the −20°C refrigerator. These rabbits were raised under standard conditions (Farm of Northwest Agriculture and Forestry University Yangling, Shaanxi, China).

Ethical Statement
All research involving animals was conducted following the Regulations for the Administration of Affairs Concerning Experimental Animals and approved by the Institutional Animal Care and Use Committee in the College of Animal Science and Technology, Northwest A&F University, Yangling, China, under permit No. DK-2019008.

Cell Experiment
The method of cell culture is consistent with that in the previous literature [23]. In short, we digested the tissue into a single cell with 0.25% collagenase type I and filtered the cells with a 40 µm cell sieve. Then, the cells were cultured at 37°C for a period of time. Finally, the induced differentiation solution (DM/F12, 0.5 mM 3-isobutyl-1-methylxanthine, 1.7 µM insulin, 10% fetal bovine serum, 1 µM dexamethasone, and 2% penicillin-streptomycin) and maintained differentiation solution (DM/F12, 1.7 mM insulin, 10% fetal bovine serum, and 2% penicillin-streptomycin) were used to differentiate the cells in the incubator (37°C, 5% CO 2 ). We used Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) to transfect the synthesis into cells.

Oil Red O Staining and Measurement of Triglyceride Content
Oil red O staining solution (Solarbio, Beijing, China) was used to stain fat droplets of adipocytes. In short, we fixed them with 4% formaldehyde for 30 min after PBS washing cells. Cells were stained in a dark environment with oil red O solution for 30 min after discarding the formaldehyde solution. After dyeing, we washed cells with water and took pictures under a microscope. Finally, 200 uL isopropanol was added and the OD value was measured at 510 nm wavelength of the microplate reader. A TG Assay Kit (Applygen, Beijing, China) was used to obtain the Intracellular triglyceride (TG) content. Firstly, the standard was prepared and diluted to obtain standards of different concentrations. Secondly, the cells were lysed using cell lysate, and the mixture was heated at 70°C for 10 min before centrifugation. Then, OD values of the supernatant and the standard samples of various concentrations were measured using the microplate reader. Finally, we calculate the content of triglyceride according to the concentration of the standard through the standard curve.

RT-qPCR
Total RNA was obtained by TRIzol Reagent (Invitrogen, Carlsbad, CA, USA). A NanoDrop 2000 spectrophotometer (Thermo, Waltham, MA, USA) was used to assess RNA quality. RT-qPCR was performed by the previous method [24] and the primers are in Table 1.

Gene-specific m 6 A qPCR
The methylation level was determined with a Magna MeRIP m 6 A Kit (Millipore, Darmstadt, Germany). Briefly, fragmented RNA binds to the antibody at 4 degrees for 12 h. After using the reaction mixture of A/G magnetic beads, we used the RNeasy kit (Qiagen, Shanghai, China) to recover the methylated RNA. Finally, the methylation level of fragmented RNA and enriched RNA was detected by qPCR.

Western Blotting
Total protein was extracted with the RIPA Lysis Buffer (CWBIO, Jiangsu, China) and Protease Inhibitor Cocktail (CWBIO, Jiangsu, China). BCA Protein Assay Kit was used to quantify the protein content. Western blotting drew on the previous literature methods [25]. The protein solution was heated at 100°C for 10 min after adding the loading buffer. Then, 40 mg proteins were added in 4-12% SDS-polyacrylamide gels for electrophoresis (1.5 h). After electrophoresis, we used a semi-dry membrane rotator to rotate the membrane for 25 min at 18 volts. The membrane was incubated for 12 h in primary antibody solution at 4°C and was incubated for 1 h in secondary antibody solution at room temperature. Finally, protein bands were obtained with a Bio-Rad GelDoc system equipped with a Universal Hood III (Bio-Rad), and a gray value was used to quantify the protein expression level.

Measurement of IMF
IMF was measured using Soxhlet petroleum-ether extraction [26]. In brief, the meat sample was chopped into minced meat, then dehydrated in an oven to a constant amount, cooled and crushed. We accurately weighed about 1.0 g of the treated sample (F) into a filter paper cylinder and then dehydrated it to a constant amount in an oven at 105°C so that the total mass was F1. Subsequently, the sample was treated in a Soxhlet extractor and was dried in an oven at 105°C to a constant amount (F2). IMF = (F1 − F2)/F × 100%.

Statistical Analysis
GraphPad Prism software 5.0 (GraphPad, Santiago, CA, USA) was used to assess all data. One-way analysis of variance (ANOVA) and two-tail Student's t-test were used in this study. A general linear model was fitted in the R software environment [27] to develop prediction models for APMAP expression level and IMF. The model is as follows, Intramuscular fat = APMAP expression level + Age. Marginal and conditional R 2 values were calculated for the model. p < 0.05 and p < 0.01 were significant and highly significant, respectively.

Methylation Modification of APMAP and APMAP Expression in Rabbits during Growth Periods
Based on previous MeRIP-seq results (the raw data are published and the results did not appear in published articles [28]), we found that mRNA of APMAP was methylated in both muscle and fat tissue ( Figure 1A). As shown in Figure 1A, the methylation of the APMAP gene in perirenal fat and muscles occurred at multiple loci and there were significant differences. The expression level of APMAP in fat and muscle increased with age ( Figure 1B,C).
Cells 2021, 10, x FOR PEER REVIEW 4 of 13 that the total mass was F1. Subsequently, the sample was treated in a Soxhlet extractor and was dried in an oven at 105 ℃ to a constant amount (F2). IMF = (F1−F2)/F×100%.

Statistical Analysis
GraphPad Prism software 5.0 (GraphPad, Santiago, CA, USA) was used to assess all data. One-way analysis of variance (ANOVA) and two-tail Student's t-test were used in this study. A general linear model was fitted in the R software environment [27] to develop prediction models for APMAP expression level and IMF. The model is as follows, Intramuscular fat = APMAP expression level + Age. Marginal and conditional R 2 values were calculated for the model. p < 0.05 and p < 0.01 were significant and highly significant, respectively.

Methylation Modification of APMAP and APMAP Expression in Rabbits During Growth Periods
Based on previous MeRIP-seq results (the raw data are published and the results did not appear in published articles [28]), we found that mRNA of APMAP was methylated in both muscle and fat tissue ( Figure 1A). As shown in Figure 1A, the methylation of the APMAP gene in perirenal fat and muscles occurred at multiple loci and there were significant differences. The expression level of APMAP in fat and muscle increased with age ( Figure 1B,C).

Preadipocytes Deletion of FTO Inhibits Adipocyte Differentiation
To investigate the role of FTO during adipogenesis, the FTO gene was interfered. The results of qPCR and WB showed that the experiment was successful (Figure 2A-C). Silencing of FTO significantly inhibited adipogenic differentiation ( Figure 2D,E). The content of Triglyceride also indicated that knockout of FTO significantly inhibited adipogenic differentiation ( Figure 2F). In addition, PPARγ C/EBPα, and FABP4 expression were inhibited when FTO was interfered ( Figure 2G-I).

Preadipocytes Deletion of FTO Inhibits Adipocyte Differentiation.
To investigate the role of FTO during adipogenesis, the FTO gene was interfered. The results of qPCR and WB showed that the experiment was successful (Figure 2A-C). Silencing of FTO significantly inhibited adipogenic differentiation ( Figure 2D,E). The content of Triglyceride also indicated that knockout of FTO significantly inhibited adipogenic differentiation ( Figure 2F). In addition, PPARγ C/EBPα, and FABP4 expression were inhibited when FTO was interfered ( Figure 2G-I).

Upregulation of FTO Increased Preadipocyte Differentiation
Tofurther make clear the role of FTO, the function studies were conducted by using pcDNA3.1 + FTO. As shown in Figure 3A-C, FTO was successfully upregulated. When

Upregulation of FTO Increased Preadipocyte Differentiation
Tofurther make clear the role of FTO, the function studies were conducted by using pcDNA3.1 + FTO. As shown in Figure 3A-C, FTO was successfully upregulated. When FTO was overexpressed, the increased lipid droplets were observed ( Figure 3D,E) and the increased content of Triglyceride was measured ( Figure 3F). In addition, adipogenic marker genes were significantly promoted ( Figure 3G-I). FTO was overexpressed, the increased lipid droplets were observed ( Figure 3D,E) and the increased content of Triglyceride was measured ( Figure 3F). In addition, adipogenic marker genes were significantly promoted ( Figure 3G-I).

The Deletion of YTHDF2 Partially Restored Adipocyte Differentiation of FTO Depleted Cells
To explore the molecular mechanisms of YTHDF2 on FTO regulating adipocyte differentiation, YTHDF2 was inhibited in FTO siRNA adipocytes. Western blot assay and qPCR were shown and our operation was effective ( Figure 4A-D). The content of triglyceride and lipid droplets was partially restored in FTO siRNA adipocytes ( Figure 4E-G). As expected, adipogenic marker gene expression levels were significantly higher than that in FTO siRNA adipocytes when YTHDF2 was knocked out (Figure 4H-J).

The Deletion of YTHDF2 Partially Restored Adipocyte Differentiation of FTO Depleted Cells
To explore the molecular mechanisms of YTHDF2 on FTO regulating adipocyte differentiation, YTHDF2 was inhibited in FTO siRNA adipocytes. Western blot assay and qPCR were shown and our operation was effective ( Figure 4A-D). The content of triglyceride and lipid droplets was partially restored in FTO siRNA adipocytes ( Figure 4E-G). As expected, adipogenic marker gene expression levels were significantly higher than that in FTO siRNA adipocytes when YTHDF2 was knocked out (Figure 4H-J).

FTO and YTHDF2 Interact to Regulate APMAP Expression
As shown in Figure 5A-C APAMP expression was significantly lower after interfering with the FTO gene (p < 0.01) whereas fold enrichment of the APMAP gene was significantly higher (p < 0.01) ( Figure 5E). As expected, overexpression of FTO achieved the opposite result ( Figure 5D). After knocking out the YTHDF2 gene in FTO siRNA adipocytes. APAMP expression was significantly higher (p < 0.01) ( Figure 5F-H) whereas fold enrichment of  ing with the FTO gene (p < 0.01) whereas fold enrichment of the APMAP gene was significantly higher (p < 0.01) ( Figure 5E). As expected, overexpression of FTO achieved the opposite result ( Figure 5D). After knocking out the YTHDF2 gene in FTO siRNA adipocytes. APAMP expression was significantly higher (p < 0.01) ( Figure 5F-H) whereas fold enrichment of methylation modification was significantly lower (p < 0.01) ( Figure 5I) than that in FTO siRNA adipocytes.

APMAP is Essential for Adipogenesis in vitro
We knocked out the APMAP gene in preadipocytes and found knockout was effective ( Figure 6A-C). Contents of triglycerides and lipid droplets indicated that APMAP could promote the differentiation of adipocytes ( Figure 6D-F). As expected, the same conclusion is drawn from the verification results of adipogenic marker genes ( Figure 6G-I).

APMAP Is Essential for Adipogenesis In Vitro
We knocked out the APMAP gene in preadipocytes and found knockout was effective ( Figure 6A-C). Contents of triglycerides and lipid droplets indicated that APMAP could promote the differentiation of adipocytes ( Figure 6D-F). As expected, the same conclusion is drawn from the verification results of adipogenic marker genes ( Figure 6G-I).

Correlation Analysis between Intramuscular Fat Content and APMAP mRNA Expression
The correlation between IMF and APMAP expression was presented in Table 2. For the model, intramuscular fat content and expression level of the APMAP gene (p < 0.01) were significant. The marginal R 2 = 0.9461 and a conditional R 2 = 0.9345 (p < 0.01). Table 2. APMAP expression affected IMF in Rex rabbits (regression coefficients, standard errors, and probability levels).

Correlation Analysis between Intramuscular Fat Content and APMAP mRNA Expression
The correlation between IMF and APMAP expression was presented in Table 2. For the model, intramuscular fat content and expression level of the APMAP gene (p < 0.01) were significant. The marginal R 2 = 0.9461 and a conditional R 2 = 0.9345 (p < 0.01).

Discussion
In recent years, scientists have been very keen on the exploration and research of fat deposition mechanisms. However, mRNA m 6 A regulates fat development poorly. In our study, we found that FTO regulated adipocyte differentiation through interference and overexpression of FTO. Previous studies have shown that FTO regulated fat deposition by promoting adipocyte differentiation [29,30]. In addition, FTO promoted the activity of FAS and PPARγ genes [31]. A previous study found that restricting feed significantly decreased FTO mRNA levels and insulin levels [32,33]. In addition, the level of IGF-1 decreased in FTO-specific deficient mice [34]. These results indicated FTO can promote insulin secretion. Studies found insulin can stimulate the phosphorylation of AKT and IRS-1 and regulate the function of AKT and IRS-1. [35,36]. Interference APMAP significantly reduced the expression of P-AKT and pIRS-1 [37]. In summary, FTO may regulate the expression level of the APMAP gene. In our study, APMAP expression decreased significantly whereas the methylation level of the APMAP gene increased significantly after transfection si-FTO ( Figure 5). These results indicated that FTO affected the expression level of the APMAP gene by m 6 A methylation.
The modification of m 6 A to mRNA transcripts should be recognized by specific proteins, which are m 6 A readers [38]. YTHDF3, YTHDF1, and YTHDF2 were identified as m 6 A readers [10,39]. YTHDF1 and YTHDF3 played an important role in protein synthesis and mRNA translation [40,41]. YTHDF2 participated in recognizing and destabilizing m 6 A-containing mRNA [10]. A previous study revealed that FTO regulated adipogenesis via m6A-YTHDF2 dependent mechanism [25]. In addition, The study also demonstrated epigallocatechin gallate targets FTO and FTO inhibited adipogenesis by YTHDF2 [42]. These results revealed that YTHDF2 can recognize FTO and regulate the expression of FTO. In our study, the data of MeRIP-seq also showed that the APMAP gene is regulated at multiple sites by m 6 A methylase ( Figure 1A). Interference of YTHDF2 partially rescued the expression level of APMAP in FTO-depleted cells (Figure 4). The methylation level of APMAP gene was significantly recovered ( Figure 5I). These results indicated FTO regulated APMAP gene expression by YTHDF2 gene.
APMAP is a regulatory factor related to adipocyte differentiation [43]. Interference APMAP inhibited adipocyte differentiation [22]. APMAP caused insulin resistance through IRS-1/insulin sensitive genes/free fatty acids pathway [44][45][46]. The activity of FAS can be improved by insulin in adipocytes [47,48] and high FAS expression significantly increased diacylglycerol deposition and caused obesity [49]. At the same time, FAS expression was positively correlated with body fat levels in many mammals [50]. In this study, we also found that APMAP promoted adipocyte differentiation ( Figure 6).
The study showed interfering with APMAP inhibited the differentiation of preadipocytes and lipid droplet formation [21]. In Table 2, we found intramuscular fat content and APMAP expression (p < 0.01) were significant. So, a high expression level of the APMAP gene means high intramuscular fat content. In addition, we found that the expression level of the APMAP gene increases with age. Studies also showed intramuscular fat content of rabbits increased with age [51]. These results indicated APMAP can regulate the intramuscular fat content of Rex rabbits. In summary, FTO regulated the expression of APMAP through YTHDF2 recognition and APMAP can promote intramuscular fat deposition (Figure 7).

Conclusion
In summary, we found FTO promoted the expression level of the APMAP gene by YTHDF2 recognition. APMAP promoted differentiation of adipocytes and APMAP expression was positively correlated with IMF content. FTO promotes intramuscular fat by targeting APMAP gene via YTHDF2 recognition ( Figure. 7). The mechanism of IMF may provide a theoretical basis for the molecular breeding of rabbits with high meat quality.

Conclusions
In summary, we found FTO promoted the expression level of the APMAP gene by YTHDF2 recognition. APMAP promoted differentiation of adipocytes and APMAP expression was positively correlated with IMF content. FTO promotes intramuscular fat by targeting.
APMAP gene via YTHDF2 recognition (Figure 7). The mechanism of IMF may provide a theoretical basis for the molecular breeding of rabbits with high meat quality.