A novel tRF-Gly is associated with obesity development through post-transcriptional regulation of lipid metabolism

Abstract

Background: Obesity, characterized by excessive fat accumulation, represents a global health crisis closely linked to metabolic disorders such as type 2 diabetes, hypertension, and atherosclerosis. tRNA-derived small RNAs (tsRNAs) have recently emerged as important epigenetic regulators, yet their roles in fat deposition remain poorly characterized. This study aims to identify tsRNAs that influence fat accumulation and to elucidate their molecular mechanisms, with a focus on tRF‑Gly‑GCC‑037 (tRF‑Gly) as a candidate regulator of adipocyte differentiation.

Methods: Visceral adipose tissue was collected from obese and lean pigs for comprehensive tRF and tiRNA sequencing. Differential expression analysis identified tRF‑Gly as a highly abundant candidate in obese samples. Functional assays in 3T3‑L1 preadipocytes included both overexpression and knockdown of tRF‑Gly, followed by lipid accumulation measurements and assessment of key adipogenic markers (CEBPα and PPARγ) by quantitative real-time PCR (qRT‑PCR) and western blot. Mechanistically, dual‑luciferase reporter assays, RNA immunoprecipitation (RIP), and nuclear-cytoplasmic protein fractionation were performed to examine how tRF‑Gly modulates the RAC1/JNK2/β‑catenin signaling axis.

Results: tRF‑Gly was significantly upregulated in visceral adipose tissue from obese pigs and ranked among the most abundant tsRNAs. Overexpression of tRF‑Gly in 3T3‑L1 cells and in C57BL/6 mice promoted lipid accumulation and increased CEBPα and PPARγ expression, whereas tRF‑Gly knockdown reduced lipid deposition. Mechanistically, tRF-Gly was suggested to bind RAC1 mRNA with AGO3 involvement, leading to RAC1 silencing. Consistently, RAC1 knockdown phenocopied the adipogenic effects of tRF-Gly, whereas RAC1 overexpression reversed these effects. Furthermore, RAC1 deficiency disrupted the RAC1/JNK2/β‑catenin complex, impaired β‑catenin nuclear translocation, and suppressed Wnt/β‑catenin signaling.

Conclusions: Our findings demonstrate that tRF‑Gly functions as a key regulator of fat accumulation. By silencing RAC1 via AGO3, tRF‑Gly disrupts RAC1/JNK2/β‑catenin complex assembly, prevents β‑catenin nuclear translocation, and downregulates Wnt/β‑catenin signaling, thereby promoting lipid deposition. This study uncovers a novel epigenetic mechanism by which tRF‑Gly controls fat accumulation and suggests that targeting tRF‑Gly may represent a therapeutic strategy for obesity and related metabolic disorders.

Keywords: Fat deposition; RAC1; RAC1/JNK2/β-catenin transport complex; Wnt/β-catenin signaling pathway; tRF-Gly.

Fig. 1

tRF-Gly expression is closely linked to lipogenesis. A VAT tissue sections from YPs and QYPs were stained using hematoxylin and eosin (H&E) (200×, 50 μm). The histogram presents the quantitative analysis of visceral adipocyte sizes in the two pig breeds. B The heatmap displays the differences in fatty acid expression of VAT between YPs and QYPs (n = 6). C The pie chart illustrates the differential metabolite types between the VAT of YPs and QYPs (n = 6). D KEGG enrichment analysis of significantly different metabolites (n = 6). E The volcano plot depicts differential tsRNA expression in VAT between YPs and QYPs, with an arrow highlighting the tRF-Gly position. F The bidirectional bar chart shows the top ten tsRNAs with the highest counts per million mapped reads (CPM) values in YPs and QYPs. G The expression of tRF-Gly in the VAT and SAT of YPs and QYPs was measured by qRT-PCR. H The cleavage site map of tRF-Gly derived from the secondary structure of tRNA-Gly. I The Venn diagram illustrates the conservation of tRF-Gly between pig and mouse species. J–L qRT-PCR of the relative expression level of tRF-Gly in mice fed either a low-fat diet (LFD) or a high-fat diet (HFD) (J), mice at different developmental stages (K), and at various differentiation time points of 3T3-L1 preadipocytes (L). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. All results above are representative of three independent experiments and presented as the means ± SEM

Fig. 2

tRF-Gly regulates 3T3-L1 preadipocytes proliferation and apoptosis. A GO enrichment analysis of tRF-Gly predicted target genes. B Evaluation of tRF-Gly transfection efficiency in proliferative phase 3T3-L1 preadipocytes using northern blot. Quantification of tRF-Gly and tRNA-Gly-GCC expression in 3T3-L1 preadipocytes by northern blot analysis, with U6 as the internal control. C The expression level of tRF-Gly in the proliferative phase of 3T3-L1 preadipocytes after transfection of the tRF-Gly mimic and inhibitor. D The Sanger sequencing of the PCR products of tRF-Gly. E The mRNA levels of proliferation and apoptosis-related genes in 3T3-L1 preadipocytes were analyzed by qRT-PCR after overexpression or knockdown of tRF-Gly. F The CCK-8 cell viability assay for the effect of tRF-Gly on the proliferation of 3T3-L1 preadipocytes. The detection time was 0, 24, 48, and 72 h after transfection. G The EdU assay for the effect of tRF-Gly overexpression or knockdown on the proliferation of 3T3-L1 preadipocytes (100×, 100 μm). H TUNEL staining showing apoptotic cells after transfection with tRF-Gly mimic or inhibitor (200× , 50 μm). I Annexin V/PI flow cytometry assay of apoptosis in 3T3-L1 preadipocytes after tRF-Gly overexpression or inhibition, with the corresponding quantification of apoptosis index. Bar graphs illustrating the percentage of viable (Q4), early apoptotic (Q3), late apoptotic (Q2), and necrotic (Q1) cells according to annexin V FITC/PI staining. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. All results above are representative of three independent experiments and presented as the means ± SEM

Fig. 3

tRF-Gly promotes lipid differentiation during the differentiation of 3T3-L1 adipocytes. A Evaluation of tRF-Gly transfection efficiency in 3T3-L1 differentiated adipocytes using northern blot. Quantification of tRF-Gly and tRNA-Gly-GCC level in 3T3-L1 adipocytes using U6 as the internal control. B tRF-Gly expression during 3T3-L1 adipocyte differentiation was analyzed following transfection with its mimic and inhibitor. C Cebpα and Pparγ mRNA expression was determined by qRT-PCR following tRF-Gly modulation. D The protein levels of CEBPα and PPARγ in 3T3-L1 adipocytes with overexpression of tRF-Gly were assessed by western blotting. The histogram quantifies the change in target protein content. E The protein levels of CEBPα and PPARγ in 3T3-L1 adipocytes with knockdown of tRF-Gly were assessed by western blotting. F The accumulation of lipid droplets in 3T3-L1 adipocytes following overexpression or knockdown of tRF-Gly was assessed by Bodipy staining. The histogram on the right quantifies the fluorescence intensity of Bodipy-stained lipid droplets (200×, 50 μm). G The accumulation of lipid droplets in 3T3-L1 adipocytes following overexpression or knockdown of tRF-Gly was assessed by Oil Red O staining. The histogram on the right quantifies the accumulation of lipid droplets stained with Oil Red O. H, I The changes in triglyceride (H) and total cholesterol (I) accumulation levels in 3T3-L1 adipocytes following overexpression or knockdown of tRF-Gly were measured using biochemical assays. J The differential changes in significant metabolites in 3T3-L1 adipocyte overexpressing tRF-Gly were analyzed by LC–MS nontargeted metabolomics. The numbers shown next to each bar indicate the corresponding Log₂ (fold change) values of metabolites (mimics tRF-Gly versus mimics NC). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. All results above are representative of three independent experiments and presented as the means ± SEM

Fig. 4

tRF-Gly is involved in the Wnt/β-catenin signaling pathway by regulating RAC1/JNK2/β-catenin transport complex expression. A KEGG enrichment analysis of tRF-Gly predicted target genes. The numbers in the bars indicate the counts of predicted target genes belonging to each pathway (e.g., 18 genes were annotated to the Wnt signaling pathway). B The chord diagram shows the ten predicted target genes of tRF-Gly associated with the Wnt/β-catenin signaling pathway. C The mRNA expression level of Rac1 in the differentiation stage of 3T3-L1 adipocytes after transfection with the tRF-Gly mimic and inhibitor. D The protein levels of RAC1 in 3T3-L1 adipocytes with overexpression or knockdown of tRF-Gly were assessed by western blotting. The histogram on the right quantifies the change in RAC1 protein content. E The luciferase activity of psiCHECK2-Rac1 was significantly decreased by the 5′ tRF-Gly mimics in 293T cells. F Schematic representation illustrating the predicted binding site of 5′ tRF-Gly with the seed region in the Rac1 3′ UTR, along with the corresponding mutation region. G The dual luciferase assay showed that interference with AGO3 expression restored the decrease in dual luciferase activity caused by the binding of tRF-Gly to psiCHECK2-Rac1. H The relationship between tRF-Gly, Rac1, and AGO3 was assessed on the basis of RIP-qPCR analysis. I Schematic diagram depicting molecular docking between tRF-Gly and AGO3. J The activation of the Wnt/β-catenin signaling pathway in 3T3-L1 adipocytes with overexpression or knockdown of tRF-Gly was assessed by western blotting. The histogram on the right quantifies the changes in target protein levels. K The nuclear translocation of β-catenin in 3T3-L1 adipocytes with overexpression or knockdown of tRF-Gly was assessed by western blotting. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. All results above are representative of three independent experiments and presented as the means ± SEM

Fig. 5

RAC1 governs the differentiation of 3T3-L1 adipocytes via the Wnt/β-catenin pathway. A The efficiency of RAC1 overexpression or knockdown was evaluated at the mRNA level by qRT-PCR. B Western blot analysis was utilized to evaluate RAC1 levels following its overexpression or knockdown in 3T3-L1 adipocytes. C The changes in adipogenic differentiation marker genes were assessed by qRT-PCR. D The changes in JNK cascade reaction marker genes were assessed by qRT-PCR. E Gene expression related to the Wnt/β-catenin pathway was measured in differentiating 3T3-L1 adipocytes following RAC1 overexpression or knockdown. F Protein expression associated with adipogenesis, JNK2, and the Wnt/β-catenin pathway was evaluated in differentiating 3T3-L1 adipocytes following RAC1 overexpression or knockdown. G The histogram shows the quantification of the immunoblotting bands from F. H Lipid droplet accumulation in differentiating 3T3-L1 adipocytes was evaluated via Bodipy staining following RAC1 overexpression or knockdown, with the accompanying histogram quantifying the fluorescence intensity. I, J The changes of triglyceride (I) and total cholesterol (J) accumulation levels in 3T3-L1 adipocytes following overexpression or knockdown of RAC1 were measured using biochemical assays. K Representative Bodipy staining of lipid droplet accumulation in 3T3-L1 adipocytes following tRF-Gly overexpression under RAC1 overexpression treatment (200×, 50 μm). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. All results above are representative of three independent experiments and presented as the means ± SEM

Fig. 6

tRF-Gly intensified high-fat-diet-induced obesity in C57BL/6 mice. A Grouping of mice injected with Agomir. The high-fat-diet-induced obesity model was established using 8-week-old C57BL/6 J mice. After a 2-week adaptation period on a high-fat diet, mice received intraperitoneal and in situ injections of Agomir-NC or Agomir-tRF-Gly (5 nmol/mouse) into iWAT and gWAT every 3 days, for a total of ten injections. B After Agomir-tRF-Gly injection, the expression levels of tRF-Gly in iWAT, gWAT, and liver were measured by qRT-PCR (n = 6 mice). C Immunohistochemistry of frozen sections showed successful enrichment of tRF-Gly in iWAT. D The line chart of body weight changes after Agomir injection in HFD, Agomir-NC, or Agomir-tRF-Gly mice (n = 6 mice). E Comparison of body size among HFD, AgomirNC, and Agomir-tRF-Gly mice (n = 6 mice). F Statistical analysis of iWAT, gWAT, and BAT adipose tissue weights in HFD, Agomir-NC, and Agomir-tRF-Gly mice. G Representative H&E-stained sections of iWAT and gWAT tissues from mice in the HFD, Agomir-NC, and Agomir-tRF-Gly groups (200×, 50 μm). The histogram presents the quantitative analysis of adipocyte size in the iWAT and gWAT of the three groups of mice. H Oil Red O staining of liver tissue from HFD, Agomir-NC, and Agomir-tRF-Gly (200×, 50 μm). The histogram shows the quantitative analysis of lipid droplet accumulation in the liver, indicating a significant increase in the Agomir-tRF-Gly group. I, J The changes in triglyceride (I) and total cholesterol (J) levels in the serum of mice from the HFD, Agomir-NC, and Agomir-tRF-Gly groups were measured using biochemical assays. The results showed significantly higher levels of TG and T-CHO in the Agomir-tRF-Gly group compared with the other groups. K The expression levels of the target genes associated with the Wnt/β-catenin signaling pathway, including Cebpα, Pparγ, Rac1, Jnk2, β-catenin, Axin1, Ck1α, and Gsk3β, were measured in the iWAT of HFD, Agomir-NC, and Agomir-tRF-Gly groups. L Western blot analysis assessed Wnt/β-catenin pathway activation in iWAT from Agomir-tRF-Gly-treated mice by measuring the levels of adipogenesis-related proteins, the JNK2/β-catenin transport complex, and degradation complex components (AXIN1, CK1α, and GSK3β). M The histogram shows the quantification of the immunoblotting bands from L. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. All results above are representative of three independent experiments and presented as the means ± SEM