Adipocyte RNA-binding protein CELF1 promotes beiging of white fat through stabilizing Dio2 mRNA

Abstract

RNA-binding proteins (RBPs) regulate diverse post-transcriptional processes and play roles in adipocyte development; however, their role in white fat beiging remains unclear. Here we identify CUG-BP Elav-like family member 1 (CELF1) as a key RBP promoting beiging of inguinal white adipose tissue in response to cold. Adipocyte-specific Celf1 deficiency impairs cold-induced thermogenic gene expression and reduces energy expenditure. Mechanistically, CELF1 binds to the 3'UTR of Dio2 mRNA and enhances its stability, promoting local triiodothyronine (T3) production. Notably, CELF1 expression is significantly reduced in subcutaneous fat of individuals with obesity and negatively correlates with BMI. CELF1 enhances isoproterenol-induced beige adipocyte activation and mitochondrial respiration in vitro, and Celf1 overexpression ameliorates diet-induced obesity and metabolic dysfunction. Hence, our study identifies CELF1 as a physiological regulator of metabolic stress in activating thermogenesis and promoting energy expenditure at the post-transcriptional level, highlighting its potential as a therapeutic target for obesity and metabolic diseases.

Fig. 1. CELF1 expression in iWAT is upregulated upon thermogenic activation.

a Venn analysis of overlapped genes between those significantly upregulated in response to cold stimulation from GSE148361 and GSE110420 (p < 0.01) and canonical RNA-binding proteins (RBPs) annotated in the RBP database. b Celf1 and Ucp1 expression in iWAT from wild-type (WT) mice exposed at 4 °C or RT for 4 days by microarray analysis from GSE148361 (n = 4 mice in each group). Celf1 (c) and Ucp1 expression (d) in iWAT from WT mice exposed at 5 °C (n = 6) or thermoneutrality (28 °C; n = 5) for 10 days by microarray analysis from GSE110420. e, f WT male mice housed at RT or 4 °C for 24 h. e qPCR analysis of Celf1 and Ucp1 mRNA levels in iWAT (n = 12 mice for RT group, n = 11 mice for 4 °C 24 h group). f Immunoblot analysis of the indicated protein in iWAT of mice (n = 4). g, h WT mice were injected with CL316,243 intraperitoneally twice, once per 12 h. g qPCR analysis of Celf1 and Ucp1 mRNA levels in iWAT (n = 7 biological replicates). h Immunoblot analysis of the indicated protein in iWAT (n = 4  biological replicates). i, j WT mice were housed at RT or 4 °C for 24 h or 7 days. i qPCR analysis of Celf1 and Ucp1 mRNA levels in iWAT of mice (n = 3 for RT and 4 °C 24 h group, n = 4 for 4 °C 7 d group). j Immunoblot analysis of the indicated protein in iWAT of mice (n = 3). Data are presented as mean ± SEM, statistical significance was determined by two-tailed unpaired Student’s t-test (b–e, g) or one-way ANOVA (i). Source data is provided as a Source Data file.

Fig. 2. Adipocyte Celf1 deficiency suppresses cold-induced iWAT beiging and energy expenditure.

a–j 8-week-old Celf1^fl/fl and Celf1^AKO mice were housed at 4 °C for 6 h. a qPCR analysis of the mRNA level of thermogenic genes in iWAT (n = 10 biological replicates). b Immunoblot analysis of the indicated protein in iWAT (n = 3 biological replicates). c Representative images of H&E staining and UCP1 immunohistochemistry (IHC) staining of iWAT, scale bar, 50 μm. VO₂ (d–e) and heat production (n = 6 mice for 22 °C group; n = 7 mice group for 4 °C) (f) of Celf1^fl/fl and Celf1^AKO mice at 22 °C and 4 °C during time (left), and average values (right) (n = 6 mice for 22 °C; n = 7 mice for 4 °C). g Rectal temperatures of Celf1^fl/fl (n = 6) and Celf1^AKO (n = 5) mice at different time points during acute cold stimulation for 6 h. Heat maps analysis of differentially expressed genes (h), Gene Ontology (GO) (i), and Kyoto Encyclopedia of Genes and Genomes (KEGG) (j) analysis of downregulated genes in iWAT of Celf1^AKO mice according to RNA-seq results. Data are presented as mean ± SEM, statistical significance was determined by two-tailed unpaired Student’s t-test (a, e, f, g). Source data is provided as a Source Data file.

Fig. 3. Adipocyte CELF1 augments isoproterenol-induced activation of beige adipocytes and mitochondrial respiration in vitro.

a–d Primary preadipocytes isolated from iWAT of WT mice were differentiated into adipocytes. qPCR (n = 6 for PBS or ISO group; n = 3 for DMSO or FSK group) or immunoblot analysis (n = 3) of CELF1 and UCP1 expression in primary adipocytes treated with PBS or ISO (a, b) and DMSO or FSK (c, d). e–k Primary adipocytes were infected with control shLacZ or shCelf1 adenovirus, control Vector or Celf1-overexpressing (Celf1) adenovirus, and then treated with PBS or ISO for 10 h before harvesting. e qPCR analysis of the mRNA level of thermogenic genes in primary adipocytes infected with shLacZ or shCelf1 adenovirus (n = 4 biological replicates). f Immunoblot analysis of the indicated protein in primary adipocytes infected with shLacZ or shCelf1 adenovirus (n = 3 biological replicates). g qPCR analysis of the mRNA level of thermogenic genes in primary adipocytes infected with Vector or Celf1-overexpressing adenovirus (n = 4 biological replicates). h Immunoblot analysis of the indicated protein in primary adipocytes infected with Vector or Celf1-overexpressing adenovirus (n = 3). i Representative analysis of OCRs of primary adipocytes infected with shLacZ or shCelf1 adenovirus in the left panel; The statistical result of basal respiration is shown in the right panel (n = 4 biological replicates). j Statistical results of maximum respiration and proton leakage respiration of panels (n = 4 biological replicates). k Representative analysis of OCRs of primary adipocytes infected with Vector or Celf1-overexpressing adenovirus in the left panel; The statistical results of basal respiration, maximum respiration, and proton leakage respiration are shown in the right panel (n = 4 biological replicates). Data are presented as mean ± SEM; statistical significance was determined by two-tailed unpaired Student’s t-test (a, c, e, g, i–k). Source data is provided as a Source Data file.

Fig. 4. CELF1 binds to the 3′UTR of mRNA and targets to thyroid hormone signaling pathway.

a Immunoblot analysis of CELF1 protein immunoprecipitated by CELF1 antibody; 10% Immunoprecipitation (IP) cell lysate was used as the input. RNA Immunoprecipitation (RIP) was conducted with differentiated primary adipocytes treated with ISO for 10 h. (Images are representative of three independent experiments) b Gene location distribution of CELF1 target according to the peak density of the RIP-Seq analysis. c Enriched sequences from the CELF1 RIP-Seq data defined by the Homer software (p < 0.05). d GO biological process analysis of CELF1 targets. e KEGG pathway enrichment analysis of CELF1 targets. f Venn analysis of overlap between differentially expressed genes from RNA-seq and enriched genes from RIP-seq. g KEGG pathway enrichment analysis of overlap genes between differentially expressed genes from RNA-seq and enriched genes from RIP-seq. h RIP-qPCR was conducted to verify target genes enriched in the thyroid hormone signaling pathway (n = 3 biological replicates). Data are presented as mean ± SEM; statistical significance was determined by two-tailed unpaired Student’s t-test (h). Source data is provided as a Source Data file.

Fig. 5. CELF1 enhances Dio2 mRNA stability via binding to its 3′UTR.

Immunoblot analysis (a) of the indicated protein in iWAT of Celf1^fl/fl and Celf1^AKO mice upon acute cold exposure and in primary adipocytes (b) of Celf1^fl/fl mice treated with PBS or ISO after adenovirus infection (n = 3). c Correlation of CELF1 and DIO2 mRNA expression in human subcutaneous adipose tissue from GSE110729 database (n = 28). RIP-qPCR analysis of the Dio2 mRNA level in IgG and CELF1 IP sample in primary adipocytes (d) and in 3T3-L1 (e) treated with ISO (n = 3 biological replicates). f RIP-qPCR analysis of Dio2 mRNA levels in CELF1 IP sample in primary adipocytes of Celf1^fl/fl mice treated with ISO after adenovirus infection (n = 3 biological replicates). g, h RNA pull-down assay and immunoblot analysis of Dio2 3’UTR bound to CELF1, β-actin CDS segment was used as a negative control, and 10% 3T3-L1 lysate was used as the input. 3’untranslated region (3’UTR) of Dio2 was divided into four segments (g), Dio2 3’UTR-1 was divided into six segments (h). qPCR analysis of Dio2 mRNA expression in primary adipocytes infected with shLacZ or shCelf1 adenovirus (n = 3 biological replicates) (i), Vector or Celf1-overexpressing adenovirus (j) following labeling with 4-thiouridine (4sU) at the indicated time points (n = 3 biological replicates). k Dual-luciferase reporting assay of Dio2 3’UTR-1 fluorescence intensity in HEK293T infected with adenovirus (n = 6 biological replicates). l, m Immunoblot analysis (l) of CELF1 immunoprecipitated by anti-CELF1 in primary adipocytes with PBS or ISO treatment. RIP-qPCR analysis (m) of Dio2 mRNA retrieved by anti-CELF1 in primary adipocytes with PBS or ISO treatment for 10 h (n = 3 biological replicates). n, o Primary adipocytes were infected with adenovirus, followed by treatment with negative control or siDio2. qPCR (n) and immunoblot analysis (o) of thermogenic genes expression after ISO treatment (n = 3 biological replicates). Data are presented as mean ± SEM; statistical significance was determined by Pearson correlation analysis (c), two-tailed unpaired Student’s t-test (d–f, i, j, m, n), and one-way ANOVA (k). Source data is provided as a Source Data file.

Fig. 6. T3 supplementation rescues Celf1-deficient induced impairment in iWAT beiging.

a–i PBS or T3 was injected into Celf1^fl/fl and Celf1^AKO male mice intraperitoneally once a day for 7 days upon cold exposure. a T3 level in iWAT (n = 7 mice for PBS group; n = 8 mice for T3 group). b Rectal temperatures detection at 2- and 7-day of cold exposure (n = 7 mice for PBS group; n = 8 mice for T3 group). Body weight (c), and iWAT weight (d) of Celf1^fl/fl and Celf1^AKO mice before and after cold exposure (n = 7 for PBS group, n = 8 for T3 group). e Representative images of H&E staining and UCP1 IHC staining of iWAT. Scale bar, 50 μm. f Immunoblot analysis of UCP1 protein in iWAT (n = 3). g qPCR analysis of thermogenic genes expression in iWAT (n = 7). VO₂ (h) and heat production (i) in PBS or T3-treated Celf1^fl/fl and Celf1^AKO mice housed at 4 °C during one 12-h light-dark cycle (left) and average values in the left panel (n = 6 for PBS group, n = 7 for T3 group). Data are presented as mean ± SEM; statistical significance was determined by two-tailed unpaired Student’s t-test (a–d, g–i). Source data is provided as a Source Data file.

Fig. 7. Adipocyte Celf1 depletion exacerbates diet-induced obesity and metabolic dysfunction.

Body weight curves (a), GTT and ITT (b) of Celf1^fl/fl and Celf1^AKO mice after ND (n = 5) or HFD (n = 6) feeding. c CELF1 mRNA expression in abdominal subcutaneous adipocytes of subjects from GSE2508 (lean, BMI = 25 ± 3; obesity, BMI = 55 ± 8; n = 9 for men, n = 10 for women). d CELF1 mRNA expression in subcutaneous adipose tissue of subjects from GSE70353 (n = 259 for BMI < 25, n = 511 for BMI > 25). e The correlation between BMI and CELF1 mRNA in subcutaneous adipose tissue of subjects from GSE70353 (n = 770). f iWAT weight of Celf1^fl/fl and Celf1^AKO mice on ND (n = 5) or HFD (n = 6). Representative images of H&E staining (g) and distribution (left) and quantification (right) of iWAT adipocyte size (h) of Celf1^fl/fl and Celf1^AKO mice under HFD (n = 6). i–s Celf1^fl/fl and Celf1^AKO mice were subjected to cold stimulation at 4 °C for 7 days after 12-week HFD feeding. Body weight loss (i) and iWAT weight (j) of HFD-fed Celf1^fl/fl and Celf1^AKO mice and after cold for 7 days (n = 9). k Representative images of H&E staining of iWAT, scale bar, 50 μm. l Rectal temperatures were monitored upon cold stimulation at the indicated time (n = 9). m qPCR analysis of thermogenic genes expression in iWAT (n = 9 biological replicates). n Immunoblot analysis of UCP1 and DIO2 protein expression in iWAT (n  =  3). o T3 concentration in iWAT of HFD-fed Celf1^fl/fl and Celf1^AKO mice after cold stimulation (n = 10). VO₂ (p), VCO₂ (q, r), and heat production (s) in HFD-fed Celf1^AKO mice and Celf1^fl/fl housed at 4 °C during one 12-h light-dark cycle (left) and average values (right) (n = 7). Data are presented as mean ± SEM; significance was assessed by Pearson correlation analysis (e), two-way ANOVA (a, b), and two-tailed unpaired Student’s t-test (c, d, f, h–j, l, m, o, p, r, s). Source data is provided as a Source Data file.

Fig. 8. Local adipocytes CELF1 overexpression in iWAT enhanced the metabolic benefits of cold stimulation in obese mice.

a Schematic of the experimental design. b–k Bilateral iWAT of male WT mice fed on high-fat diet for 7 weeks were injected with Adipoq-promoter driven control AAV virus (AAV-Vector) and Celf1-overexpression AAV virus (AAV-Celf1) for four weeks, followed by cold stimulation for one week. b Rectal temperatures of AAV-Vector and AAV-Celf1 mice at different time points during cold stimulation (n = 6). c Body weight of AAV-Vector and AAV-Celf1 mice before and after cold stimulation (n = 6). d iWAT weight of AAV-Vector and AAV-Celf1 mice (n = 6). e Representative images of H&E staining and UCP1 IHC staining of iWAT from AAV-Vector and AAV-Celf1 mice, scale bar, 100 μm. qPCR analysis of thermogenic genes expression (f), T3 levels (g), VO₂ (h), and heat production (i) of AAV-Vector and AAV-Celf1 mice housed at 4 °C during one 12-h light-dark cycle (left) and average data (right) (n = 6). GTT (j) and ITT (k) of AAV-Vector and AAV-Celf1 mice (n = 10). l Schematic of the experimental design. m–t Bilateral iWAT of 7-week-old ob/ob male mice was injected with Adipoq-promoter driven Celf1 overexpression and control AAV virus, after 4 weeks, the mice were exposed to cold stimulation at 15 °C for a week (n = 6). Body weight curve (m) and weight loss (n) of ob/ob mice after AAV-Vector and AAV-Celf1 infection. o Rectal temperatures of AAV-Vector and AAV-Celf1 ob/ob mice at different time points during cold stimulation (n = 6). p iWAT weight of AAV-Vector and AAV-Celf1 ob/ob mice (n = 6). q qPCR analysis of thermogenic genes expression in iWAT of AAV-Vector and AAV-Celf1 ob/ob mice (n  = 5 biological replicates). T3 concentration in iWAT (r), GTT (s), and ITT (t) of AAV-Vector and AAV-Celf1 ob/ob mice (n = 6). Data are presented as mean ± SEM; statistical significance was determined by two-tailed unpaired Student’s t-test. Source data is provided as a Source Data file.