Interference of a mammalian circRNA regulates lipid metabolism reprogramming by targeting miR-24-3p/Igf2/PI3K-AKT-mTOR and Igf2bp2/Ucp1 axis

Abstract

White adipose tissue (WAT) is important for regulating the whole systemic energy homeostasis. Excessive WAT accumulation further contributes to the development of obesity and obesity-related illnesses. More detailed mechanisms for WAT lipid metabolism reprogramming, however, are still elusive. Here, we report the abnormally high expression of a circular RNA (circRNA) mmu_circ_0001874 in the WAT and liver of mice with obesity. mmu_circ_0001874 interference achieved using a specific adeno-associated virus infects target tissues, down-regulating lipid accumulation in the obesity mice WAT, and liver tissues. Mechanistically, miR-24-3p directly interacts with the lipid metabolism effect of mmu_circ_0001874 and participates in adipogenesis and lipid accumulation by targeting Igf2/PI3K-AKT-mTOR axis. Moreover, mmu_circ_0001874 binds to Igf2bp2 to interact with Ucp1, up-regulating Ucp1 translation and increasing thermogenesis to decrease lipid accumulation. In conclusion, our data highlight a physiological role for circRNA in lipid metabolism reprogramming and suggest mmu_circ_0001874/miR-24-3p/Igf2/PI3K-AKT-mTOR and mmu_circ_0001874/Igf2bp2/Ucp1 axis may represent a potential mechanism for controlling lipid accumulation in obesity.

Supplementary Information

The online version contains supplementary material available at 10.1007/s00018-023-04899-1.

Introduction

Obesity is visualized as an excess of white adipose tissue (WAT) accumulation leading to escalating effects on health, including fatty liver, cardiovascular risk, and even cancer [1–3]. The incidence of obesity has increased rapidly over the past 20 years, reaching epidemic levels, and it is increasing at an even faster rate among teenagers [4]. Obesity is a complex lipid dysmetabolism condition, and foods that are readily available, affordable, highly appealing, and heavy in fat represent an increased risk for the development of obesity [5, 6]. To prevent or suppress obesity, the maintenance of lipid metabolism equilibrium is crucial [7]. WAT is a multifunctional organ supporting triglyceride (TG) metabolism as well as the release of inflammatory cytokines and adipokines [8]. Moreover, WAT is the first tissue affected by obesity, which depends on the unique ability of the adipocytes to coordinate metabolic changes across the body and to integrate responses to maintain metabolic homeostasis [8, 9]. When the chronic nutritional intake is overwhelmed, the lipogenesis process is accelerated, which further contribute to the hypertrophy of mature adipocytes. These modifications have an impact on the WAT microenvironment such as inflammation and the interaction between WAT and other organs [10]. For instance, when TG is improperly stored in adipocytes and free fatty acid concentrations are greater than normal, the liver function to maintain the homeostasis of lipid metabolic activity is disrupted [11]. Importantly, most adipose tissue-derived molecules such as inflammatory cytokines and adipokines are alternated, which are critical for interorgan crosstalk and contribute to the process of fatty liver [9].

Circular RNA (circRNA) and microRNA (miRNA) are two intrinsic components that are important for lipid metabolism reprogramming. Among these, circRNA is produced from the host gene through a non-canonical splicing event known as back-splicing with neither 5ʹ–3ʹ polarity nor a polyadenylated tail [12]. However, circRNA, a non-linear RNA, also works through protein sponges, miRNA sponges, and even coding functions. Besides, miRNA is a small RNA molecule that regulates target gene function by pairing to the 3′-UTR to direct their post-transcriptional repression. Similarly, circRNA and miRNA are highly tissue specific, species conserving, and developmental specific. Numerous studies indicated that circRNA and miRNA have a role in lipid metabolism and lipid disorders-related illnesses. circRNA and miRNA exhibited altered expression patterns in adipocytes or liver tissue during obesity [13–15]. Functionally, circFUT10/let-7c/PPARGC1B axis is crucial for controlling cattle adipocytes proliferation and differentiation [16]. circSAMD4A and circTshz2-1 facilitated adipogenesis whereas circPTK2 accelerated adipolysis in the fat tissue [17–19]. In hepatocytes, circRNA_0000660 silencing aggravated lipid metabolism, whereas circScd1 over-expression suppressed the synthesis of lipid droplets [20, 21]. Moreover, over-expression of circRNA_0001805 reduced the lipid buildup in the liver and played a synergistic role against non-alcoholic fatty liver disease-induced lipid metabolism disorder [22].

Overview, circRNA and miRNA have attracted much interest for their role in lipid metabolism and illnesses. Even though major pathways leading to lipid accumulation in adipocytes or hepatocytes have been discovered, there are many functions of circRNA and miRNA awaiting further exploration in the context of obesity. From the data made available online, we identified a particular miRNA miR-24-3p with roles in myogenesis [23], cancer [24], Alzheimer's disease [25], and many other processes such as adipogenesis [26] and diabetes [27]. In addition, miR-24-3p is linked to mitophagy, which is a potential mechanism for an indirect effect on these disparate functions [28]. It is interesting to note that miR-24-3p abundance reduced in rats plasma [29], blunt snout bream liver [30], and pig fat tissue [31] following high-fat diet treatment or adipose again. We identified a circRNA called mmu_circ_0001874, which is associated with miR-24-3p and examined their function in vitro and vivo. Mechanistically, mmu_circ_0001874 interacts with lipid metabolism by targeting miR-24-3p/Igf2/PI3K-AKT-mTOR axis. Additionally, mmu_circ_0001874 binds to Igf2bp2 to interact with Ucp1, up-regulating Ucp1 translation and increasing thermogenesis to decrease lipid synthesis. Our data indicate that mmu_circ_0001874/miR-24-3p/Igf2/PI3K-AKT-mTOR and mmu_circ_0001874/Igf2bp2/Ucp1 axis may play an important role in the regulation of lipid metabolism reprogramming and may contribute to the development of new therapies for obesity.

Materials and methods

Animals

In this work, rabbits primary preadipocytes were collected from the newborn Tianfu black Rabbits perirenal area using the method described earlier [32]. Besides, eighteen 21-day male Kunming mice were randomly divided into three groups. Among these, two groups of mice received either a normal diet (marked as CON) or a high-fat diet (marked as HFD) for three weeks and received a total of four tail vein injections of agomir control (RiboBio, Guangzhou, China). The remaining group received a high-fat diet and four tail vein injections of miR-24-3p agomir (HFD-A; RiboBio, Guangzhou, China) over three weeks. Besides, to study the function of mmu_circ_0001874, two groups of 21-day male Kunming mice were tail vein injected with 100 μL control adeno-associated virus (AAV) and given either a normal diet (designated as CON-2) or a high-fat diet (designated as HFD-2) for 4 weeks. One group of mice fed a high-fat diet was tail vein injected with 100 μL eGFP-labeled AAV-9 designed for interfering mmu_circ_0001874 (HFD-A2; 10¹² vg/mL, Hanbio Tech, Shanghai, China) for 4 weeks. In addition, identical three groups of mice were fed a normal diet or a high-fat diet for 14 weeks and injected with 100 μL control AAV or eGFP-labeled AAV-9 at 8 weeks (once every 3 weeks; normal diet + control AAV: CON-3; high-fat diet + control AAV: HFD-3; high-fat diet + eGFP-labeled AAV-9: HFD-A3). Table S1 displays the nutritional breakdown of the normal diet and high-fat diet. The number of mice was 6 for each group. All mice were given free access to food and water. Animals were slaughtered humanely to minimize suffering when the trial was complete. The experimental procedures were approved by the Animal Care and Use Committee from the College of Animal Science and Technology, Sichuan Agricultural University, China.

Cell culture and transfection

Cells were kept in the growth medium (GM) containing 10% fetal bovine serum (FBS; ExCell Bio, Shanghai, China) in an incubator (Thermo Fisher Scientific, San Jose, CA, USA) at 37 °C and 5% CO₂ environment. For cells differentiation experiments, after 2 days of cells contact inhibition, the medium of primary preadipocytes or 3T3-L1 cells was changed to the differentiation medium (DM) containing Dulbecco's modified Eagle's medium (DMEM; VivaCell, Shanghai, China) supplemented with 10% FBS, 1 μmol/L dexamethasone (DEX; Solarbio, Beijing, China), 0.5 mmol/L 3-isobutyl-1-methylxanthine (IBMX; Solarbio, Beijing, China), and 10 μg/mL insulin (Solarbio, Beijing, China) for 3 days. After that, the DM was changed to the maintenance medium (MM) consisting of DMEM with 10% FBS and 10 μg/mL insulin for 3 days. Subsequently, the medium was replaced with GM to get mature adipocytes. Cell transfection was performed using the lipofectamine 3000 reagent (Invitrogen, Carlsbad, CA, USA), adhering to the manufacturer's instructions. The transfection reagent was mixed with RNA oligo [miR-24-3p mimic, inhibitor, negative control (NC), inhibitor negative control (INC)], siRNA of Igf2 [si-Igf2-1, si-Igf2-2, si-Igf2-3, and related normal group (si-Igf2-NG)], siRNA of mmu_circ_0001874 [si-circ-1, si-circ-2, and related normal group (si-circ-NG)], siRNA of Igf2bp2 and related normal group (si-Igf2bp2-NG), or plasmids [pcDNA3.1-Igf2 and empty plasmid (EMP)], and then transfected into the cells. The above RNA oligo was purchased from Sangon Biotech Co., Ltd (Shanghai, China). siRNA and plasmids were purchased from Tsingke Biotechnology Co., Ltd (Beijing, China). The sequence information is shown in Table S2-A.

Establish the hypertrophic cells model

Palmitate (PA) is a physiological component of TG in adipocytes. Here, we introduced the serum-free medium with PA (Sigma, Shanghai, China) for the aim of paring the hypertrophic cells model [33]. Briefly, 4 mmol PA was added to 0.1 mol/L NaOH (50 mL) for 30 min at 70 °C and then mixed with 10% bovine serum albumin (BSA; Sigma, Shanghai, China) to get the 1 mmol/L PA mather liquor. Finally, the mother liquor was diluted with the serum-free medium and filtered to obtain a 0.3 mmol/L PA working solution. For the blank group (BC), the mother liquor was replaced with the same volume of 10% BSA and mixed with the serum-free medium. At 48 h after the addition of PA or BC, cells were collected for further trial.

RNA fluorescence in situ hybridization (FISH)

The information about the specific probe used in FISH for mmu_circ_0001874 and miR-24-3p is shown in Table S2-B. The tissue sample was prepared according to the FISH kit's recommended procedure (BersinBio, Guangzhou, China), and then it was incubated with the probe at 37 °C for 16 h. The nuclei were stained with DAPI. Finally, images were captured using an inverted fluorescent microscope from Olympus in Tokyo, Japan.

Quantitative real-time polymerase chain reaction (qRT-PCR)

Following the instructions, total RNA was extracted using the Trizol Reagent (Qualityard Bio, Beijing, China). An Agilent Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) was used for the quality test, and only eligible RNA was used for the later trial. The RT Easy™ II (With gDNase) (FOREGENE, Chengdu, China) and the Mir-X™ miRNA First-Strand Synthesis Kit (Takara, Dalian, China), respectively, for reverse transcription of mRNA and miRNA. In addition, qRT-PCR was carried out in triplicate using the abm^® EvaGREEN 2× qPCR Master Mix (ABM Inc., Canada) on a CFX96 instrument (Bio-Rad, Hercules, CA, USA), and the relative abundance was calculated using the 2^−ΔΔCt method. The mRQ 3′ primer in the Mir-X™ miRNA First-Strand Synthesis Kit (Takara, Dalian, China) served as a reverse primer for miRNA quantification, and U6 was used as an internal reference. Besides, β-actin was used as the internal reference for mRNA quantification. Table S2-C lists the sequence information in detail.

Western blotting (WB)

Using the ProteinExt^® Mammalian Total Protein Extraction Kit (TransGen Biotech, Beijing, China) and Nuclear Protein Extraction Kit (Solarbio, Beijing, China), respectively, total protein and nuclear protein were extracted. Next, the concentration of protein was tested using the Bradford protein assay kit (Novoprotein, Shanghai, China), and only protein meeting the criteria (concentration > 10 mg/mL) was used for further trial. Briefly, the denatured protein was resolved on the SurePAGE gels (GenScript Corporation, Nanjing, China) and then transferred to a PVDF membrane, followed by sealing of the sealing fluid. The membranes were first incubated for 8 h at 4 °C with the matching primary antibodies, then with the secondary antibodies [Goat Anti-Rabbit or Anti-Mouse IgG H&L (HRP), Zen bioscience, Chengdu, China] for 1 h. The antibodies information is as follows: Igf2bp2 (11601-1-AP), Ucp1 (23673-1-AP), PCNA (10205-2-AP), and LPIN1 (27026-1-AP) were purchased from the Proteintech (Wuhan, China); Fsp27 (bs-6796R) was purchased from the Bioss (Beijing, China); PPARG (A0270), CEBPA (A0904), FABP4 (A0232), and SCD (A16429) were purchased from the Abclonal (Wuhan, China); and mTOR (380411), P-mTOR (381557), AKT (382804), P-AKT (310021), PI3K (251221), SREBP1 (347061), IL-1β (511369), TNF-α (346654), β-actin (380624), Igf2 (820814), FASN (31582), and IL-6 (347023) were purchased from Zen bioscience (Chengdu, China). The membranes were subjected to an ECL chemiluminescence reagent (HAKATA, Shanghai, China) to detect immunoreactivities. A GelDoc system equipped (Bio-Rad, Hercules, CA, USA) was used to capture images.

Identification of circRNA ring formation

Following the manufacturer's instructions, the DNA sample was extracted using a commercial StarSpin Universal DNA kit (Genstar, Beijing, China). cDNA was collected using the above-mentioned method. Next, a PCR trial was performed using the 2× Spark Taq Plus PCR Master Mix (with dye) (Shandong Sparkjade Biotechnology Co., Ltd, China). Table S2-C contains a list of the divergent and convergent primer sequence information. Then, the PCR product was identified by Nucleic acid electrophoresis and followed by Sanger sequencing at Tsingke Biotechnology Co., Ltd (Beijing, China). RNase R (4U, 40 U/L) from the GENESEED Co., Ltd. (Guangzhou, China), together with 10× reaction buffer (2 μL) and suitable water (up to 20 μL), was incubated with one component (2 μg RNA) for 10 min at 37 °C. A second portion (2 μg RNA) was incubated at 37 °C for 10 min with 10× reaction buffer (2 μL) and appropriate DEPC water (up to 20 μL). Next, the abundance of circRNA was identified by reverse transcription, PCR amplification, and nucleic acid electrophoresis.

Staining

The cells were washed with PBS and then fixed in 4% paraformaldehyde for 30 min at room temperature. Next, Oil Red O (Servicebio, Wuhan, China) was mixed with deionized water at a rate of 3:2 and then added to the cells for 15 min. Finally, the above cells were rinsed with PBS until there were no obvious contaminants. Similarly, we can observe the lipid content of the liver under the light microscope after the cryosection was stained with Oil Red O. In addition, animal fat and liver tissues were stained with hematoxylin–eosin (HE) to examine the histomorphological changes. Firstly, the samples were fixed in 4% paraformaldehyde for 24 h and then washed with sterile water. Secondly, the specimens were successively dehydrated and embedded in paraffin, followed by HE staining. Finally, a microtome (RM2235, Leica, Nussloch, Germany) was used to get the 5-µm-thick sections, and images were captured using an inverted microscope (Olympus, Tokyo, Japan). The adipocytes area was calculated using the image j program using the scale plate as a size reference. The specified antibodies were used in immunohistofluorescence staining at Wuhan Servicebio Technology Co., Ltd.

RNA immunoprecipitation (RIP)

The PureBinding^® RNA Immunoprecipitation Kit (GENESEED, Guangzhou, China) was used following the instructions to perform the RIP analysis. Briefly, specific Igf2bp2/IMP2 rabbit pAb (Proteintech, Wuhan, China) and rabbit pAb control IgG (Abclonal, Wuhan, China) antibodies, respectively, reacted with magnetic beads reacted at 4 °C for 2 h and then incubated with 3T3-L1 cells clarified lysate with rotation at 4 °C overnight. qRT-PCR and WB analyses were then performed on the RNA and protein on the beads, respectively. The input group utilized was clarified lysate.

Glucose tolerance test (GTT) and insulin tolerance test (ITT)

GTT and ITT were performed after overnight fasting when the diet therapy was complete. The mice received an intraperitoneal injection of glucose (1.5 g/kg, Sigma, Shanghai, China) or insulin (0.5 IU/kg, Wanbang Biopharma, Jiangsu, Ching) solution provided as a mother liquor in water. The blood sample was collected by tail bleeding at 0, 15, 30, 60, and 120 min after injection. Finally, a glucose analyzer (Sinocare, Changsha, China) was used to measure the blood glucose.

Measure of blood fat and enzyme-linked immunosorbent assay (ELISA) analysis

All mice were fasted overnight before slaughter and anesthetized via intraperitoneal injection of chloral hydrate. The blood was collected from the heart and then centrifuged at 4 °C for 5 min to get the serum. Using the commercial kit based on enzymatic colorimetry (Jiancheng, Nanjing, China), the serum concentration of TG, cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) were each measured following the standard guidelines. Tissues or cells were gathered, dissected, and their TG content was checked using the aforementioned kit. Besides, following the standard method, the rabbits (Shanghai Hengyuan biotechnology Co., Ltd, China) and mice (Quanzhou Ruixin biotechnology Co., Ltd, China) commercial ELISA kit were employed and the leptin, adiponectin, IL-6, IL-1β, and TNF-α were measured, respectively.

Measure of cells proliferation

Transfection was carried out when the cell density reached 50%. After 6 h, the medium was changed to GM. Cell counting kit (CCK; Zomanbio, Beijing, China) and 5-Ethynyl-2ʹ-deoxyuridine (EDU; RiboBio, Guangzhou, China) analysis were performed to examine the effect of miR-24-3p on cells proliferation. Briefly, after 0, 1, and 2 days of transfection, 10 μL CCK reagent was added to each well and incubated at 37 °C and 5% CO₂ for 2 h. After that, a microplate reader (Thermo Fisher Scientific, USA) was used to measure the absorbance, which reflects the number of live cells. Additionally, we chose the 48-h-transfected cells for the EDU analysis. Detailly, the cells were cultured for 2 h in GM containing 100 μL EDU. Next, the cells were immobilized and stained according to the company's instructions. The staining of EDU and Hoechst in the same field was photographed using an inverted fluorescence microscope (Olympus, Tokyo, Japan). Images were analyzed by using image-pro plus 6.0 software (Media Cybernetics, Inc, Rockville, MD, USA).

Dual-luciferase reporter assay

Here, we predicted the potential miR-24-3p targets using the database starbase (https://starbase.sysu.edu.cn/). Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were performed using the software DAVID 6.7 (http://david.abcc.ncifcrf.gov/home.jsp). The RNA22 website (https://cm.jefferson.edu/rna22/Interactive/) was used to determine the probable binding site between miRNA and mRNA or circRNA based on the sequencing data, respectively. To validate the predicted site, luciferase reporter plasmids [wild-type (WT) and mutant of target sequence] were constructed by Tsingke Biotechnology Co., Ltd (Beijing, China). Next, 293 T cells were seeded into 24-well plates (NEST Biotechnology, Jiangsu, China). The miR-24-3p mimic or NC was co-transfected with specific WT or mutant plasmids using the lipofectamine 3000 reagent (Invitrogen, Carlsbad, CA, USA) when the cell density reached 70%. Following the manufacturer's instructions, luciferase activities were then assessed using the Duo-Lite TM Luciferase Assay System (Vazyme, Nanjing, China) after 24 h.

Transcriptome sequencing

Sample RNA was prepared using the procedure mentioned earlier. The first strand of cDNA was synthesized in the M-MuLv reverse transcriptase system, and then the second strand of cDNA was synthesized using the dNTPs and DNA polymerase I. After repair, adding the poly (A) tail, and connection sequencing connector, cDNA of 250–300 bp was generated and then PCR amplificated to build the cDNA libraries using the NEBNext^® Ultra™ RNA Library Prep Kit for Illumina^® (New England BioLabs, Ipswich, MA, USA), following the manufacturer's method. Finally, Novogene Co., Ltd. (Beijing, China) sequenced the cDNA libraries using the Illumina NovaSeq 6000 platform. Additionally, the raw data were cleaned with the FASTP program (version 0.19.7). The clean data were then compared with the reference genome using HISAT2 software to obtain the localization information of reads. For circRNA, the circbase database was used as a reference. After that, each sample's transcript read count was calculated, and the DEseq package from R was used for differential expression (DE) analysis. The threshold was defined to be |log2(fold change)| ≥ 1, and p-value ≤ 0.05. The DE genes between groups were visually shown using the R ggplot2 package and the function Heatmap.

Statistical analysis

All data are presented as means ± SEM. GraphPad software (GraphPad Software Inc., La Jolla, CA) was used for statistical analysis, and differences between groups were determined by Student's t test. Moreover, multiple comparisons were performed using one-way or two-way ANOVA analysis. At a p-value < 0.05, differences were deemed statistically significant.

Results

miR-24-3p is associated with lipid metabolism

miRNA is highly conserved among species and exhibits specific temporal and spatial expression patterns in response to various biological processes [34, 35]. To further verify miR-24-3p expression in different obese animal models, we measured its abundance in the perirenal fat and liver samples from normal and obese rabbits obtained in our prior study [36], and a decreased miR-24-3p abundance was validated, consistent with published data [29–31] (Fig. 1A). We measured the levels of the hormones leptin and adiponectin, which are secreted by the WAT, in the serum of rabbits. In the perirenal fat, miR-24-3p expression was negatively connected with leptin levels but positively correlated with adiponectin levels (Fig. 1B). In the liver, it was confirmed that TG from obese rabbits had a negative association with miR-24-3p abundance (Fig. 1C). Besides, preadipocytes differentiation is a critical process in adipogenesis and is characterized by the dynamic expression of multiple miRNAs [8]. Studies on adipogenesis in rabbits preadipocytes from newborn rabbits indicated that miR-24-3p maintained a relatively low abundance after differentiation but gradually up-regulated at the late stage of differentiation (Fig. 1D, E). We also confirmed the abundance of miR-24-3p was decreased in rabbits mature adipocytes with PA treatment (Fig. 1F).

Fig. 1.

miR-24-3p is associated with lipid metabolism. A Schematic representation of rabbits model underwent a control or a high-fat diet for 5 weeks in an environmentally controlled room (21–23 °C, 60–75% humidity, 14-h light [60 lx]). miR-24-3p expression was measured in the rabbits perirenal fat and liver tissues, and the data in the normal rabbits were a reference (n = 6). B The Pearson correlation between miR-24-3p expression in the rabbits perirenal fat tissue and serum leptin (left) or adiponectin (right) levels (n = 12 in biological animals). C The Pearson correlation between liver TG levels and miR-24-3p expression pattern in the liver tissue of rabbits (n = 12 in biological animals). D After the rabbits preadipocytes were differentiated in vitro on days 0, 2, 4, 6, and 8, Oil Red O staining was performed for visual observation of lipid droplets. E miR-24-3p expression in rabbit preadipocytes was examined using qRT-PCR during the differentiation process (n = 6). F miR-24-3p was decreased in rabbits mature adipocytes under PA treatment (n = 6). G The top 5 gene sets enriched in the KEGG database were listed. Blank group: BC; Palmitate: PA. E, F three samples from each group were used for qRT-PCR analysis and two duplicate samples were measured. The data are presented as means ± SEM. *p-value < 0.05; **p-value < 0.01

Furthermore, we used the starbase database to get 2547 genes that are putative miR-24-3p target genes, including Igf2, Cish, and Tk1 (Table S3-A), to better understand the biological function of miR-24-3p from a bioinformatics viewpoint. The starbase database presented the miRNA–target interactions by intersecting the predicting target sites of miRNA with binding sites of Ago protein [37]. As determined by the GO analysis, miR-24-3p target genes were significantly enriched in 450 GO terms [250 biological processes (BP), 106 cellular components (CC), and 94 molecular functions (MF)], including negative regulation of cell growth, regulation of cell proliferation, regulation of cell differentiation, and lipid storage (Table S3-B). miR-24-3p target genes have enhanced activities in KEGG analysis that were closely connected to metabolic processes, cancer disorders, etc. (Table S3-C). The top 5 gene sets were enriched in pathways in cancer, PI3K-AKT signaling pathway, endocytosis, MAPK signaling pathway, and Ras signaling pathway (Fig. 1G). Together, our findings suggest that miR-24-3p may have multiple biological functions and be involved in lipid metabolism.

miR-24-3p regulates rabbits preadipocytes proliferation and differentiation

Moreover, we further performed CCK and EDU analysis to assess the impact of miR-24-3p on rabbits preadipocytes proliferation. After 2 days of transfection, transfection efficiency was measured in the context of proliferation. miR-24-3p expression was observed at higher levels in the mimic group compared to the NC group, but miR-24-3p expression in the inhibitor group was significantly lower than those in the INC group, demonstrating that transfection analogs were successful in boosting or lowering miR-24-3p abundance in vitro (Fig. 2A). Meanwhile, miR-24-3p mimic of various lengths of time all decreased the absorbance, a marker of cells proliferation, to different extents, but miR-24-3p inhibitor significantly increased the absorbance of rabbits preadipocytes after transfection 2 days (Fig. 2B). The EDU trial revealed, as shown in Fig. 2C, D, that the number of positive cells was lower in the miR-24-3p mimic group than in the NC group, while it was higher in the inhibitor group than in the INC group. Moreover, we confirmed the similar alternations of DNA replication-relative protein PCNA for all tested candidates (Fig. 2E, F, Fig. S1A).

Fig. 2.

miR-24-3p regulates rabbits preadipocytes proliferation and differentiation. A Transfection efficiency detection of miR-24-3p mimic and inhibitor in the context of proliferation (n = 9). B The absorbance of rabbits preadipocytes at 0, 1, and 2 days after transfection with miR-24-3p mimic, NC, miR-24-3p inhibitor, and INC (n = 5). C The percent of EDU-positive cells [(EDU-positive cells rate = EDU-positive cells/Hoechst-stained cells) × 100%] (n = 3). D The picture of the EDU proliferation assay for rabbits preadipocytes transfected with miR-24-3p mimic, NC, miR-24-3p inhibitor, and INC (red fluorescence represents EDU-positive cells, and nuclei are indicated by blue fluorescence). (E–F, n = 3 in F). Representative bands for PCNA in rabbits preadipocytes transfected with miR-24-3p mimic, NC, miR-24-3p inhibitor, and INC 2 days in the context of proliferation. G Transfection efficiency detection of miR-24-3p mimic and inhibitor in the context of differentiation (n = 9). Representative WB of PPARG, CEBPA, and FABP4 and their quantitative analysis at 2 days (H, I) and 8 days (J, K) after transfection with miR-24-3p mimic, NC, miR-24-3p inhibitor, and INC in rabbits preadipocytes in the context of differentiation. n = 3 in I and K. Three samples from each group were used for qRT-PCR analysis and three duplicate samples were measured. The data are presented as means ± SEM. *p-value < 0.05; **p-value < 0.01

Next, after 2 days of cells contact inhibition (designated as the 0 day of differentiation), transfection was carried out and the transfection effectiveness was assessed after 2 days of transfection in the setting of differentiation (Fig. 2G). Meanwhile, we discovered an obvious and highly significant decrease (increase) of proteins in the miR-24-3p mimic (inhibitor) group, including PPARG, CEBPA, and FABP4, which are crucial transcription factors in the development and function of fat tissue and markers of preadipocytes differentiation (Fig. 2H, I, Fig. S1B). At the late stage of differentiation (8 days), quantification of PPARG, CEBPA, and FABP4 immunoblotting performed in the mimic group showed a consistent decrease relative to the NC group, which were reversed upon miR-24-3p inhibitor (Fig. 2J, K, Fig. S1C). In conclusion, these data emphasize the indispensable role of miR-24-3p against rabbits preadipocytes proliferation and differentiation in vitro.

In vivo over-expression of miR-24-3p resistant to high-fat diet-induced obesity

Next, we investigated the physiological consequences of over-expression miR-24-3p in vivo. The miR-24-3p over-expression model was established, whereas mice were injected with miR-24-3p agomir and given a high-fat diet. We first confirmed that fat and liver tissues showed a significant difference in miR-24-3p abundance between the HFD and HFD-A groups (Fig. 3A). At weeks 2 and 3, the high-fat diet treatment significantly raised body weight; however, miR-24-3p agomir prevented the development of high-fat diet-induced body weight gain (Fig. 3B). The HFD group had significantly higher levels of the two serum fat indexes, TG and TC, which are indicators of lipid metabolism and obesity, while the HFD-A group had lower levels of TG and TC related to the HFD group (Fig. 3C). Additionally, we concentrated on contrasting the weight of fat and liver tissues between groups. Under high-fat diet condition, mice with miR-24-3p over-expression showed a retarded tissue weight gain (Fig. 3D). A histological analysis revealed that the HFD-A group had adipocytes with smaller cell areas and reduced lipid buildup in the liver compared to the HFD group overall (Fig. 3E–G). Meanwhile, inflammation factor IL-6 in the HFD-A group fat and liver tissues was retarded (Fig. 3F). To further measure the role of miR-24-3p in high-fat diet-induced obesity, GTT and ITT were performed, and the data indicated that miR-24-3p agomir treatment decreased the blood glucose values after intraperitoneal injection of glucose or insulin (Fig. 3H, I). Moreover, we compared the molecular levels of lipogenesis-related genes FASN and SREBP1 and pro-inflammatory genes IL-8, IL-1β, and TNF-α in the perirenal fat between groups. Interestingly, on a high-fat diet, their expression was consistently reduced following miR-24-3p agomir therapy (Fig. 3J). We confirmed some similar changes at the protein levels (Fig. 3K, L, Fig. S2A). The major genes involved in the TG biosynthesis pathway in the liver, Agpat1, Agpat3, and Mogat1, were up-regulated in the liver following a high-fat diet treatment, while miR-24-3p agomir slow rate was detected [38] (Fig. 3M). Fsp27 displayed comparable alternations between groups, which is a protein that localizes to lipid droplets and promotes TG formation [39] (Fig. 3N, O, Fig. S2B). These findings suggest that active miR-24-3p over-expression prevents the development of obesity caused by a high-fat diet via lowering lipid accumulation in vivo.

Fig. 3.

In vivo over-expression of miR-24-3p resistant to high-fat diet-induced obesity. (A) miR-24-3p expression was identified by qRT-PCR analysis in the perirenal fat and liver tissues from the 42 days CON, HFD, and HFD-A mice (n = 9). Analysis of metabolic performances of CON, HFD, and HFD-A groups was described, including body weight (B), serum parameters TG and TC (C), fat and liver tissues weight (D), representative HE staining of fat and liver tissues (E, F), immunohistofluorescence for IL-6 in the fat and liver tissues (F), liver TG levels (G), GTT (H), ITT (I), mRNA levels of lipogenesis-related genes FASN and SREBP1 and pro-inflammatory genes IL-8, IL-1β, and TNF-α in the perirenal fat (J), immunoblotting for FASN, SREBP1, IL-1β, Igf2, TNF-α, and β-actin in the perirenal fat (K, L), mRNA levels of marker genes involved in the liver TG biosynthesis in the liver (M), immunoblotting for Fsp27, Igf2, and β-actin in the liver (N, O). n = 6 in B–D, G–I; n = 25 in E; n = 9 in J and M; n = 3 in L and O. Three samples from each group were used for qRT-PCR analysis and three duplicate samples were measured. The data are presented as means ± SEM. *p-value < 0.05; **p-value < 0.01

As a miR-24-3p target, Igf2 is required for lipogenesis via PI3K-AKT-mTOR pathway

To further assess the molecular implications of miR-24-3p over-expression in vivo, we next measured changes in mRNA expression by sequencing in the same perirenal fat in which we observed miR-24-3p dysregulation. Overview, ten libraries were built successfully and the summary and quality assessment of the mRNA sequencing data are listed in Table S4-A. Genes expression was quantified using the subread software, and DE genes (129 up and 119 down) were identified using the DESeq following the standard of |log2(fold change)| ≥ 1 and p-value ≤ 0.05 (Fig. 4A, Table S4-B). DE genes with similar expression levels across different samples were grouped to obtain an overview of DE genes (Fig. 4B). KEGG-GSEA analysis indicated deregulated pathways, which are lipogenesis-related pathways such as the PI3K-AKT signaling pathway and mTOR signaling pathway (Fig. 4C), and diseases-related pathways such as Alzheimer disease and breast cancer, as well as cell metabolism such as apoptosis, cell cycle, and osteoclast differentiation. Moreover, we cross-analyzed the predicted target genes at the starbase database with mRNA sequencing data from the perirenal fat tissue and focused on the intersection of genes involved in metabolic processes. A total of 12 genes (Igf2, Fam20c, Tk1, Ptprt, Asf1b, Plxna4, Gfod1, Themis, Kcnj11, Itk, Cish, Unc5c) were identified, and we were intrigued by the discovery of Igf2 as a crucial possibility, a functional gene connected to adipose biology, lipid metabolism, and obesity, as well as a potential miR-24-3p target.

Fig. 4.

As a miR-24-3p target, Igf2 is required for lipogenesis via PI3K-AKT-mTOR pathway. A A volcano picture of markedly changed genes between the HFD and HFD-A groups was built based on log2(fold change) and -log10(p-value). The statistically significant up-regulated genes are shown by the red point, and the statistically significant down-regulated genes are represented by the green point. B Hierarchical clustering analysis of DE genes in the HFD-A group compared with the HFD group (red module represents up-regulated genes and blue module represents down-regulated genes). C KEGG-GSEA analysis was applied for pathway analysis. D The predicted binding site between gene Igf2 and miR-24-3p. In the CON, HFD, and HFD-A groups perirenal fat and liver, the expression levels of Igf2 at mRNA (E, n = 9) levels were tested. F Luciferase assays were performed by co-transfection of Igf2 WT and mutant plasmids with miR-24-3p mimic and NC, respectively, in 293 T cells, and the WT + NC group was used as the control group (n = 3). G, H After suppression expression and over-expression of Igf2 gene, the Igf2 protein abundance was examined using WB. I mRNA of FASN, SREBP1, IL-1β, and TNF-α in 3T3-L1 differentiated mature adipocytes were detected using qRT-PCR (n = 9). J, K Extracted protein of 3T3-L1 differentiated mature adipocytes treated with siRNA or over-expression of Igf2 was immunoblotted for FASN, SREBP1, IL-1β, PI3K, AKT, P-AKT, mTOR, P-mTOR, and β-actin. L qRT-PCR analysis of Agpat1, Agpat3, and Mogat1 in hepatocytes treated with siRNA or over-expression of Igf2 (n = 9). M, N WB analysis measured the protein abundance of Fsp27 in hepatocytes treated with siRNA or over-expression of Igf2. O, P Nucleus LPIN1 abundance was tested using the nucleus protein extracted from 3T3-L1 differentiated mature adipocytes treated with suppression expression and over-expression of Igf2 gene. Three samples from each group were used for qRT-PCR analysis and three duplicate samples were measured. Blank group: BC; Palmitate: PA. The data are presented as means ± SEM. *p-value < 0.05; **p-value < 0.01

The RNA22 website estimated the probable binding site between Igf2 and miR-24-3p (Fig. 4D). Igf2 expression was shown to be up-regulated in the perirenal fat and liver of the HFD group, but it was found to be down-regulated after miR-24-3p over-expression (Fig. 3K, L, Fig. S2A, Fig. 3N, O, Fig. S2B, Fig. 4E). Using the luciferase reporter analysis, we verified the aforementioned hypothesis. miR-24-3p mimic significantly decreased the luciferase activity of a luc-reporter construct containing Igf2 3ʹ UTR (WT), while mutant of the Igf2 binding site in the 3ʹ UTR abrogated this reduction (Fig. 4F). Next, we delivered specific siRNA targeting Igf2 and over-expression plasmids of Igf2 into 3T3-L1 cells. After 48 h, Igf2 protein dropped after knockdown in cells, particularly in the si-Igf2-2 group, but increased following plasmid administration (Fig. 4G, H, Fig. S3A). The 3T3-L1 mature adipocytes and hepatocytes were then transfected with si-Igf2-2 and pcDNA3.1-Igf2, respectively, to test the impact of Igf2 on lipid metabolism. The expression levels of FASN, SREBP1, IL-1β, and TNF-α in 3T3-L1 differentiated mature adipocytes were enhanced after PA treatment for 48 h, but PA + si-Igf2-2 group showed a reduced expression of the above molecules related to the PA + si-Igf2-NG group and vice versa in the PA + pcDNA3.1-Igf2 group when compared with the PA + EMP group and further WB validation of FASN, SREBP1, and IL-1β proteins (Fig. 4I–K, Fig. S3B). The mRNA Agpat1, Agpat3, and Mogat1, as well as the protein Fsp27, were both down-regulated in hepatocytes to comparable levels in the PA + si-Igf2-2 group but up-regulated in the PA + pcDNA3.1-Igf2 group (Fig. 4L–N, Fig. S3C). In addition, intracellular TG analysis showed siRNA of Igf2 reduced lipid accumulation, while over-expression Igf2 increased lipid accumulation following PA treatment (Fig. S4A-B).

In addition, we further wanted to determine whether Igf2 functions in the regulation of lipid metabolism via the PI3K-AKT-mTOR pathway. We first confirmed that Igf2 siRNA along with over-expression Igf2 had a significant impact on PI3K activity in 3T3-L1 differentiated mature adipocytes (Fig. 4J, K, Fig. S3B). Then, siRNA of Igf2 suppressed P-AKT expression, whereas over-expression Igf2 increased the P-AKT expression, concurrent with no significant effect on the total AKT abundance (Fig. 4J, K, Fig. S3B). The downstream mTOR showed a reduction in the PA + si-Igf2-2 group but drastically increased in the PA + pcDNA3.1-Igf2 group when compared with the related reference, while the phosphorylated mTOR showed a similar alternation (Fig. 4J, K, Fig. S3B). As a phosphatidic acid phosphatase, nucleus LPIN1 induces lipogenesis-related transcription factor activity and showed an opposite abundance with the mTOR (Fig. 4O, P, Fig. S3D). In conclusion, our data indicate that Igf2 is a direct miR-24-3p target and interaction with the lipid metabolism effect via the PI3K-AKT-mTOR pathway.

mmu_circ_0001874 acts as a sponge of miR-24-3p

Given that miR-24-3p has a lower abundance in the high-fat diet-fed animal fat and liver tissues, we think about whether a circRNA acts as a ceRNA sponge to interact with miR-24-3p and affect the development of obesity. To test this hypothesis, we sequenced circRNA in the perirenal fat of six mice (three CON and three HFD). We obtained an average of 113.0417 M high-quality clean reads per sample (range 112.06–114.59 M) with clean reads ratio ≥ 98.78%, and the clean reads were then aligned to reference genomes using the HISAT, with high mapping efficiency of ≥ 94.18% (Table S5-A). Additionally, circRNA abundance was measured using the read count approach, and 174 DE circRNAs (59 up-regulated and 115 down-regulated) was identified based on the |log2(fold change)| ≥ 1 and p-value ≤ 0.05 using the DESeq (Table S5-B). To discover the critical circRNA that regulates miR-24-3p and may be involved in the process of lipid metabolism, we concentrated on the up-regulated circRNA. Detailly, 22 circRNAs had sites that binding to miR-24-3p using the RNA22 website, and only 4 circRNAs (mmu_circ_0001874, mmu_circ_0003575, mmu_circ_0016413, and mmu_circ_0005513) had 2 or more miR-24-3p binding sites (Fig. 5A). mmu_circ_0001874 had the most, containing 5 sites, and high significance and abundance, which caught our interest. Finally, mmu_circ_0001874 derived from the 6720401G13Rik gene (also known as Firre) on chromosome X was screened as the potential miR-24-3p upstream regulator for the subsequent experiment. The expected head-to-tail junction was amplified in the cDNA and validated in line with the sequence information of mmu_circ_0001874 by Sanger sequencing (Fig. 5B). Following RNase R treatment, it was discovered that mmu_circ_0001874 is not digestible by RNase R (Fig. 5C). Then, we verified that the abundance of mmu_circ_0001874 was raised in the HFD fat and liver demonstrating the reliability of the sequencing results, and also improved following PA therapy at the cellular level (Fig. 5D). Sequence analysis indicated the existence of specific miR-24-3p binding site on mmu_circ_0001874. Based on the potential binding sites for dual-luciferase reporter analysis, we created mmu_circ_0001874 mutant1 (including site1 to site4), mutant2 (including site5), and related WT1 and WT2, and the results indicated that the luciferase activity was significantly decreased in the WT1 + mimic and WT2 + mimic groups but not significantly changed in the mutant1 + mimic and mutant2 + mimic groups when compared with the related control (Fig. 5E, F). We also discovered that miR-24-3p and mmu_circ_0001874 were co-localized in the cytoplasm using the RNA FISH analysis (Fig. 5G). Taken together, mmu_circ_0001874 is identified as a stable circRNA interacting with miR-24-3p in the mice fat and liver tissues.

Fig. 5.

mmu_circ_0001874 acts as a sponge of miR-24-3p. A The flow chart showed the screening rules of circRNA that may regulate miR-24-3p. B Divergent primer (< >) and convergent primer (> <) amplified mmu_circ_0001874 in cDNA, but only convergent primer amplified mmu_circ_0001874 in gDNA. The amplified product of divergent primer was consistent with the sequence information by Sanger sequencing. C mmu_circ_0001874 was amplified with divergent primer (< >) in both RNase R+ and RNase R− groups. D mmu_circ_0001874 expression was enhanced in the HFD perirenal fat and liver, and was enhanced in 3T3-L1 differentiated mature adipocytes after treatment with PA (n = 9). E Using the RNA22 website, five potential binding sites between mmu_circ_0001874 and miR-24-3p were identified based on the sequence information. F The relative luciferase activity was measured after co-transfection of mmu_circ_0001874 WT1, WT2, mutant1, and mutant2 with miR-24-3p mimic and NC, respectively (n = 3). G mmu_circ_0001874 and miR-24-3p’s cellular localization was identified utilizing the particular probe. Three samples from each group were used for qRT-PCR analysis and three duplicate samples were measured. Blank group: BC; Palmitate: PA. The data are presented as means ± SEM. *p-value < 0.05; **p-value < 0.01

Lack of mmu_circ_0001874 decreases lipid accumulation in vitro and in vivo

Two distinct siRNAs against the mmu_circ_0001874 back-splice sequence were designed, and the effectiveness of the interference was assessed. As shown in Fig. 6A, mmu_circ_0001874 abundance was strongly down-regulated in the si-circ-2 group, with less than a doubling of mmu_circ_0001874 abundance in the si-circ-NG group. Consequently, si-circ-2 was chosen for the later trial. We discovered that si-circ-2 prevented TG accumulation in 3T3-L1 differentiated mature adipocytes and hepatocytes under PA condition in vitro by measuring the total intracellular TG (Fig. S4C). Additionally, lipid droplet alternations were seen in conjunction with the reported effect of TG accumulation (Fig. S4D). According to these findings, mmu_circ_0001874 may be essential for lipid metabolism in adipocytes and hepatocytes.

Fig. 6.

Lack of mmu_circ_0001874 decreases lipid accumulation in vitro and vivo. (A) In 3T3-L1, the interfering efficiency of mmu_circ_0001874-related siRNA was measured by qRT-PCR analysis (n = 9). (B) Mice in vivo fluorescence imaging analysis revealed eGFP-labeled AAV construct was specific infected perirenal fat and liver tissues and mmu_circ_0001874 and miR-24-3p levels were observed by qRT-PCR analysis (n = 9). Analysis of metabolic performances of CON-2, HFD-2, and HFD-A2 groups was described, including body weight (C), fat and liver tissues weight (D), serum TG abundance (E), representative HE staining of fat and liver tissues and Oil Red O staining for lipid droplets in the liver (F, J), liver TG levels (G), GTT (H), ITT (I), and lipid metabolism-related proteins and pro-inflammatory-related proteins levels in the perirenal fat (K, L) and liver (M, N), respectively. n = 6 in C–E, G–I; n = 25 in F; n = 3 in L and N. Three samples from each group were used for qRT-PCR analysis and three duplicate samples were measured. The data are presented as means ± SEM. *p-value < 0.05; **p-value < 0.01

We subsequently set out to assess the physiological interaction between mmu_circ_0001874 and lipid metabolism in vivo. We established a fat and liver-specific mmu_circ_0001874 inhibition of function model by administrating the experimental AAV construct or control AAV in the mice model. Fluorescence imaging analysis was used to locate the AAV construct, and the effective interfering efficiency was calculated, indicating that the AAV construct specifically inhibited mmu_circ_0001874 in both fat and liver tissues (Fig. 6B). Importantly, down-expressed mmu_circ_0001874 having the enhanced effect on miR-24-3p expression (Fig. 6B). The animals with mmu_circ_0001874 inhibition (HFD-A2 group) specifically showed a reduction in body weight, fat tissue weight, and serum TG levels compared to the HFD-2 group when placed on a high-fat diet (Fig. 6C–E). They also had smaller adipocytes and decreased hepatic steatosis, along with reduced TG levels and lipid droplets (Fig. 6F, G, Fig. 6J). Notably, compared to the HFD-2 group, the HFD-A2 group exhibited consistently lower blood glucose values after intraperitoneal injection of glucose or insulin (Fig. 6H, I). In addition, after consuming a high-fat diet, we noticed that mice with experimental AAV construct had opposite lipogenesis-related protein abundances compared to mice with control AAV (Fig. 6K, L, Fig. S5A). Additionally, we found that mmu_circ_0001874 liver inhibition reduced the abundance of Fsp27 in the HFD-A2 group compared to the HFD-2 group (Fig. 6M, N, Fig. S5B).

Thinking from a therapeutic point, we applied the AAV construct on chronic high-fat diet-induced obese mice to measure its effects in treating obesity. Before administering the experimental AAV construct or control AAV, mice were given a high-fat diet for 8 weeks to induce chronically obese mice. Interestingly, the prolonged treatment caused obese mice to experience similar lipid metabolic benefits of mmu_circ_0001874, including decreased body weight, fat tissue weight, liver TG, blood TG, TC, and LDL-C, and raised serum HDL-C levels compared to their control (Fig. S6A-D). Therefore, these data suggest that mmu_circ_0001874 interference may both prevent and treat obesity and metabolic dysfunctions.

mmu_circ_0001874 deficiency protects obesity-induced inflammation

Hypertrophic adipocytes further promote the inflammatory response in the fat tissue as well as in the liver in an environment with abundant energy [10]. Leptin, an essential adipokine released by the WAT is a pro-inflammatory factor, whereas adiponectin is an anti-inflammatory adipokine. Compared to the HFD-2 group, the blood leptin concentration was lower, while adiponectin levels were higher in the HFD-A2 group (Fig. 7A). Additionally, when the HFD-2 groups were utilized as a control, the IL-6, IL-1β, and TNF-α in the serum samples decreased in the HFD-A2 group (Fig. 7B). Interestingly, the perirenal fat and liver IL-6, IL-1β, and TNF-α levels also showed similar alternations with the levels tested in the serum samples (Fig. 7B). Pro-inflammatory genes such as IL-6, IL-1β, TNF-α, IL-8, Cd11c, MCP1, iNOS, and Ifng had higher mRNA levels in the perirenal fat tissue in the HFD-2 group (related to the CON-2 group), whereas they had lower levels in the animals injected with the experimental AAV construct (HFD-A2 group, related to the HFD-2 group), and some markers were further confirmed at protein levels as determined by WB and immunohistofluorescence trial (Fig. 6K, L, Fig. S5A, Fig. 7C, D). Similarly, inhibition of mmu_circ_0001874 in the liver decreased the pro-inflammatory genes-related protein levels by WB and immunohistofluorescence trials (Fig. 6M, N, Fig. S5B, Fig. 7D). Additionally, in mice given a high-fat diet for 14 weeks, the effects of mmu_circ_0001874 under the chronically obese state with inflammation were studied. As expected, mmu_circ_0001874 inhibition of function not only showed the effects of reducing body and tissue weight and controlling blood lipid in already obese individuals (see “Lack of mmu_circ_0001874 decreases lipid accumulation in vitro and vivo”) but also demonstrated improved anti-inflammatory activity. Serum leptin levels decreased between 14 weeks of high-fat diet mice receiving experimental AAV construct or control AAV, whereas adiponectin levels changed in the opposite direction (Fig. S6E). Pro-inflammatory markers (IL-6, IL-1β, and TNF-α), ELISA of serum, perirenal fat, and liver showed a similar enhanced pattern in the HFD-3 group when compared with the data in the CON-3, while IL-6, IL-1β, and TNF-α were decreased in the HFD-A3 group related to the HFD-3 group, and immunohistofluorescence confirmed that the abundance of IL-6, IL-1β, and TNF-α was decreased in the high-fat diet-induced obese mice with mmu_circ_0001874 inhibition of function (Fig. S6F-G). Thus, our data indicate that mmu_circ_0001874 deficiency has anti-inflammatory effects on both fat and liver tissue in vivo.

Fig. 7.

mmu_circ_0001874 deficiency protects obesity-induced inflammation. A The CON-2, HFD-2, and HFD-A2 mice serum leptin and adiponectin levels were measured using the ELISA kit (n = 6). B ELISA analysis revealed the serum IL-6, IL-1β, and TNF-α levels from the CON-2, HFD-2, and HFD-A2 groups (n = 6). C The relative mRNA abundance of pro-inflammatory genes in the CON-2, HFD-2, and HFD-A2 groups perirenal fat was measured (n = 12). D Expression of IL-6, IL-1β, and TNF-α was observed in the CON-2, HFD-2, and HFD-A2 mice perirenal fat and liver tissues by immunohistofluorescence trial. Six samples from each group were used for qRT-PCR analysis and two duplicate samples were measured. The data are presented as means ± SEM. *p-value < 0.05; **p-value < 0.01

mmu_circ_0001874 directly binds to Igf2bp2 and functions through thermogenesis

circRNA has multiple biological functions in lipid metabolism regulation. circRNA can serve as direct RNA-binding proteins (RBP) sponges in addition to sponging miRNA [40]. Predicted analysis was carried out for mmu_circ_0001874 utilizing the RBPsuite [41] and catRAPID [42] databases to look into this possibility. The data indicated that multiple proteins were identified as the potential mmu_circ_0001874-interacting proteins from the above databases (Table S6-A-B). Interestingly, we noted that proteins Igf2bp1, Igf2bp2, and Igf2bp3 are candidates identified in both RBPsuite and catRAPID databases, which caught our attention. Based on the Ct value, measurement of endogenous Igf2bp1, Igf2bp2, and Igf2bp3 mRNA levels showed that Igf2bp2 had higher abundance (data not shown). Igf2bp2 may bind mmu_circ_0001874 at multiple sites (Fig. 8A, B). In vivo, we discovered that a high-fat diet increased Igf2bp2 abundance, which was then reversed when mmu_circ_0001874 deficit occurred (Fig. 8C–E, Fig. S7A). Combined FISH and immunofluorescence staining demonstrated strong co-localization signals between mmu_circ_0001874 and Igf2bp2 in the adipocytes cytoplasm (Fig. 8F). RIP trial indicated that mmu_circ_0001874 co-precipitated with endogenous Igf2bp2 protein (Fig. 8G).

Fig. 8.

mmu_circ_0001874 directly binds to Igf2bp2 and functions through thermogenesis. The Igf2bp2 motif was analyzed using the RBPsuite (A) and catRAPID (B) databases to screen for bind regions of mmu_circ_0001874 that Igf2bp2 is most likely to bind to. C The relative Igf2bp2 mRNA expression was tested in the CON-2, HFD-2, and HFD-A2 groups perirenal fat (n = 6). The CON-2, HFD-2, and HFD-A2 groups perirenal fat underwent immunoblotting to detect the proteins Igf2bp2 and Ucp1 (D), and their expression was quantitatively analyzed (E, n = 3). F FISH staining of mmu_circ_0001874 followed by immunofluorescence staining of Igf2bp2 protein and nucleus staining with DAPI. G mmu_circ_0001874 was enriched by Igf2bp2 in 3T3-L1 cells. n = 6 for qRT-PCR. H In the CON-2, HFD-2, and HFD-A2 groups perirenal fat, the relative mRNA levels of thermogenic and mitochondrial genes were examined (n = 6). I The relative TG content was measured in 3T3-L1 cells treated with si-Igf2bp2-NG and si-Igf2bp2 (n = 6). J qRT-PCR analysis verified the transfection efficiency in 3T3-L1 cells treated with si-Igf2bp2, si-circ-2, and relative control (n = 6). K, L Representative bands for Igf2bp2 and Ucp1 in 3T3-L1 cells treated with si-Igf2bp2, si-circ-2, and relative control. n = 3 in L. Three samples from each group were used for qRT-PCR analysis and two duplicate samples were measured. The data are presented as means ± SEM. *p-value < 0.05; **p-value < 0.01

Igf2bp2 was enriched in mRNA binding, mRNA transport, energy homeostasis, negative regulation of translation, and other GO terms (Table S6-C). Thus, we next wanted to determine whether Igf2bp2 is functionally involved in mmu_circ_0001874-induced lipid metabolism by regulating gene expression. Here, we found that mmu_circ_0001874 deficiency increased the mRNA levels of thermogenic and mitochondrial genes as well as Ucp1 protein but had no effect on the signaling for lipolysis in the fat tissue of obese mice (Fig. 8D, H). After Igf2bp2 gene silencing, the 3T3-L1 cells had less lipid accumulation, according to intracellular TG concentration (Fig. 8I). Moreover, mmu_circ_0001874 deficiency in 3T3-L1 cells resulted in a decrease in Igf2bp2 mRNA levels, but Igf2bp2 gene silencing did not affect the mmu_circ_0001874 expression, indicating that Igf2bp2 does not modulate the stability of mmu_circ_0001874 but may interact with mmu_circ_0001874 to regulate the lipid metabolism (Fig. 8J). Photoactivatable ribonucleoside-enhanced CLIP (PAR-CLIP) sequencing analysis showed the existence of multiple Igf2bp2 target mRNA [43]. Among these, the thermogenesis gene Ucp1 is also an Igf2bp2 mRNA client, and evidence indicated that Igf2bp2 binds Ucp1 mRNA and inhibits its translation [44]. We, therefore, consider whether mmu_circ_0001874 might be necessary for Igf2bp2-mediated post-transcriptional regulation of its target Ucp1. To achieve this, we co-transfected the cells with si-circ-2 and si-Igf2bp2, and the data indicated that mmu_circ_0001874 deficiency significantly boosted Igf2bp2 gene silencing-induced Ucp1 expression (Fig. 8K, L, Fig. S7B). Thus, our results suggest that mmu_circ_0001874 binds to Igf2bp2 and may be vital for Ucp1-induced thermogenesis.

Discussion

Excessive WAT accumulation is linked to unbalanced energy homeostasis and obesity-related illnesses such as inflammation and fatty liver. Thus, it would be worthwhile to understand the molecular mechanism and network of lipid metabolism, including miRNA and circRNA capable of effective lipid metabolism control to inhibit lipid accumulation in the WAT and liver. In this study, we established the miR-24-3p and mmu_circ_00001874 dysfunction mice models using the specific miRNA agomir and AAV construct, respectively. Our data indicate that mmu_circ_0001874/miR-24-3p/Igf2/PI3K-AKT-mTOR and mmu_circ_0001874/Igf2bp2/Ucp1 axis are crucial for protecting against the development of high-fat diet or PA-induced lipid metabolism disorder as well as related complication.

Mature adipocytes are derived from the differentiation of preadipocytes and are capable of lipid synthesis, storage, and production of adiponectin and leptin [45]. Preadipocytes comprise 15–50% of all cells in the white fat depots [46]. Preadipocytes' rapid succession of proliferative and differentiating processes leads to the growth of adipose tissue and weight gain. miRNA is an endogenous small RNA that plays a significant role in animals by pairing to the mRNA of protein-coding genes to direct their post-transcriptional repression. Numerous studies have shown that miRNA, important players in the control of metabolism, functions in adipogenesis. For instance, 3T3-L1 preadipocytes express miR-214-3p, which acts through transcription factor Ctnnb1 and WNT/β-catenin signaling pathway [47]. miR-9-5p over-expression causes the development of mature adipocytes with lipogenic features by up-regulating transcription factors FABP4 and PPARG [32]. miR-24-3p was reported to have inhibitory action during the proliferation of N87 cells [48], mesangial cells [49], human skin fibroblasts [50], and during differentiation of human periodontal ligament stem cells [51], and human adipose-derived mesenchymal stem cells [52]. However, miR-24-3p boosted proliferation while prevented differentiation of intramuscular preadipocytes in chickens by suppressing ANXA6 [53]. Here, miR-24-3p maintained a relatively low abundance after differentiation but gradually up-regulated at the late stage of differentiation in preadipocytes, inverse with the observed enhancement of transcription factors FABP4, CEBPA, and PPARG [32]. We hypothesized that miR-24-3p may offer an “unlocking” mechanism that functions to engage with distinct signals under differentiated circumstances within the preadipocytes to maintain adipogenesis by releasing the transcription factors FABP4, CEBPA, and PPARG. It will be intriguing to investigate these possibilities, but the dearth of relevant in vivo and vitro models, the technical challenges in performing effective and conditional over-expression or knockdown of miR-24-3p, and the complicated phenotypes observed will likely require the cooperation of many signals.

Importantly, boosting the expression of miR-24-3p in the obesity mice perirenal fat and liver is beneficial for maintaining lipid metabolic homeostasis by sponging endogenous Igf2, and their interaction supports earlier research [54]. Our results indicate that over-expression of miR-24-3p alters high-fat diet-induced body weight gain, tissue weight gain, blood lipid levels, adipocytes size, liver TG levels, glucose tolerance and insulin tolerance, and inflammation in the obesity mice. Previous reports showed that Igf2 plays a central role in gluconeogenic control, lipid metabolism, and insulin signaling pathways and is necessary for maintaining glucose homeostasis as well as the activation of the gluconeogenic regulatory program [55, 56]. Igf2 is reported to inhibit lipid accumulation and inflammatory responses in macrophages [57], and its genetic variants are associated with obesity and obesity-related hypertension [58]. Upon binding to the type I IGF receptor, Igf2 triggers the receptor tyrosine kinase activity, which leads to phosphorylation of itself and its major substrate, the insulin receptor substrate 1 (IRS-1), and further activates the PI3K/AKT cascades, and depending on the cell type, stimulates proliferation, differentiation, or both [59]. Here, we found that Igf2 is direct interaction with the lipogenesis effect of miR-24-3p via the PI3K-AKT-mTOR pathway in 3T3-L1 differentiated mature adipocytes. In addition, in the miR-24-3p over-expression investigation carried out in the fat tissue, we discovered an enrichment of down-regulated miR-24-3p targets, some of which, such as MAPK7 [60], PHB2 [61], Bcl-2L11 [62], and Igfbp5 [63], are validated miR-24-3p targets. Meanwhile, genes SEMA3G, Dact1, and Cidea, which are known regulators of lipid accumulation, were also decreased. In any case, activation of them has been liked to lipid accumulation, which can be induced by both cell-extrinsic and cell-intrinsic signals. SEMA3G knockdown was found to inhibit weight gain, reduce fat mass, prevent lipogenesis in the liver tissue, reduce insulin resistance, and ameliorate glucose tolerance in obesity mice [64]. Dact1 regulates adipogenesis through coordinated effects on gene expression that selectively alters intracellular and paracrine/autocrine components of the Wnt/beta-catenin signaling pathway [65]. Cidea plays a critical role in promoting hepatic lipid accumulation and in the development of hepatic steatosis by acting as a sensor that responds to diets that contain fatty acids [66]. Thus, the results demonstrate that miR-24-3p is crucial for controlling lipid metabolism, though we cannot deny the exist of potential extra non-Igf2 effects due to ectopic expression of miR-24-3p in vivo study. Besides, it would be necessary to investigate the roles of miR-24-3p in other animal models in the process of lipid metabolism, as well as the other potential mechanisms of miR-24-3p on lipid metabolism, including fat browning, enhanced lipid secretion through the skin, metabolomics, will be necessary to elucidate these issues.

circRNA transcribed from thousands of loci across the human and mouse genomes, many of which are species conserved and tissue specific, exhibits a particular pattern of down-regulation in the fat tissue during obesity [13]. In line with this notion, we identified 174 DE circRNAs using high-throughput RNA-seq analysis between the CON and HFD mice fat tissue, of which 59 circRNAs were up-regulated and 115 circRNAs were down-regulated. It is speculated that these circRNAs maintain equilibrium under the normal status of lipid metabolism. We do not know how this equilibrium is disturbed during the development of lipid disorder diseases and whether the disequilibrium is a cause or a result of lipid disorder diseases. However, there is no doubt that a distinct pattern between various study groups exists due to the experiment background [18]. It is probable that adipocytes underwent significant remodeling as a result of obesity, and circRNA displays dynamic modulation in hypertrophic adipocytes and is essential for the development of lipid disorders. Here, it is of great interest to identify a circRNA termed mmu_circ_00001874 that functions as a ceRNA sponge to interact with miR-24-3p and supports lipid metabolism. Our data firstly demonstrate the vivo role of mmu_circ_00001874 in driving the transcriptional program that supports core metabolism pathways such as TG metabolism and biosynthesis and inflammation by targeting endogenous miR-24-3p, resulting in adjustable Igf2 expression. It is worth noting that no discernible change was observed in the liver weight of mice with mmu_circ_00001874 manipulation compared to control mice on a high-fat diet, but fat tissues exhibited a bigger difference. This discrepancy suggest the possible role of mmu_circ_00001874 in the fat and other metabolic tissues and the exist of metabolic differences among these tissues. Given that mmu_circ_00001874 is encoded on chromosome X, it would also be intriguing to investigate the relationship between mmu_circ_00001874 and sex-associated traits as well as the differences in fat deposition between the sexes.

Another possibility is that mmu_circ_00001874 contributes to thermogenesis in the mice WAT with obesity via enhancing Ucp1 translation by binding to RBP Igf2bp2. Igf2bp2, named for their ability to bind to the leader 5ʹ and 3ʹ UTR of Igf2 mRNA, coinciding with the expression of Igf2 polypeptide. Global elimination of Igf2bp2 animals are highly resistant to high-fat diet-induced obesity and fatty liver and display superior glucose tolerance and insulin sensitivity, increased energy expenditure, and greater core temperature defense against cold exposure [44]. Liver-specific over-expression of Igf2bp2 causes steatosis [67], while Igf2bp2 deletion in the liver reduces hepatic fatty acid oxidation [68]. Moreover, beige or brite (brown-in-white) adipocytes are present in the WAT and have a white fat-like phenotype that when stimulated acquires a brown fat-like phenotype, increasing thermogenesis, and this phenomenon is referred to as browning. The best-characterized thermogenic effector is Ucp1 protein. Ucp1 localizes to the mitochondrial inner membrane. Ucp1 knockout mice are unable to maintain their body temperature and develop hypothermia upon acute cold challenge [69]. According to earlier research, Igf2bp2 binds the mRNA encoding Ucp1 and other mitochondrial components, and most of them translate more effectively without Igf2bp2 [44]. Our data discover that since mmu_circ_00001874 and Igf2bp2 are both located in the cytoplasm, mmu_circ_00001874 may regulate the translation of Ucp1 protein by binding to Igf2bp2, indicating the potential connections between mmu_circ_00001874 and metabolic network and may provide new insight into the mechanism underlying circRNA functions. However, we cannot exclude that mmu_circ_00001874 may cooperate with other acts through unidentified ways to control lipid metabolism. As a result, more in-depth mechanistic research is required to elucidate how mmu_circ_00001874 controls lipid metabolism reprogramming.

Our findings imply that mmu_circ_0001874/miR-24-3p/Igf2/PI3K-AKT-mTOR axis and mmu_circ_0001874/Igf2bp2/Ucp1 axis are crucial for lipid metabolism reprogramming (Fig. 9). Down-expressed mmu_circ_0001874 reduced TG accumulation and inflammation, and improved miR-24-3p transcription and abnormal glucose tolerance. We discovered that the ratio of mmu_circ_0001874 to other regulatory factors such as miR-24-3p and Igf2bp2 changes dynamically with metabolic stimuli such as a high-fat diet (Fig. 9) and that mmu_circ_0001874 decrease will be a promising diagnostic and therapeutic target in lipid metabolism reprogramming, bringing a new dimension to the functional importance of circRNA regulation in obesity. In addition, it would be intriguing to further understand the other potential mechanisms for the beneficial effects of mmu_circ_0001874 would lead to a more thorough understanding. Future research is necessary to identify the mmu_circ_0001874 function human homolog, comprehend the mechanism, and decipher the direction of the interaction.

Fig. 9.

mmu_circ_0001874 is an important regulator in lipid metabolism reprogramming via miR-24-3p/Igf2/PI3K-AKT-mTOR and Igf2bp2/Ucp1 axis and is a possible therapeutic target for obesity-related metabolic illnesses

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

The project was planned and designed by JS, JW, XJ, and SL. Data were gathered and research was carried out by JS, MW, ZL, GJ, and TT. JS and AZ wrote this paper.

Funding

The National Modern Agricultural Industrial Technology System (CARS-43-A-2) and the Key Research and Development Program of Sichuan Province (2021YFYZ0033) provided funding for our research.

Availability of data and materials

The sequencing data are available on NCBI database at Sequence Read Archive (SRA). The transcriptome sequencing data: SUB12222924.

Declarations

Conflict of interest

There is no conflict of interest to declare.

Consent for publication

Not applicable.

Ethics approval and consent to participate

The experimental procedures were approved by the Animal Care and Use Committee from the College of Animal Science and Technology, Sichuan Agricultural University, China.