Adipocyte microRNA-802 promotes adipose tissue inflammation and insulin resistance by modulating macrophages in obesity
- State Key Laboratory of Natural Medicines, Jiangsu Key Laboratory of Druggability of Biopharmaceuticals, School of life Science and Technology, China Pharmaceutical University, China
- NanJing HanKai Academy, China
- State Key Laboratory of Natural Medicines, China Pharmaceutical University, China
- Jiangsu Key Laboratory of TCM Evaluation and Translational Research, School of Traditional Chinese Pharmacy, China Pharmaceutical University, China
Figures
Figure 1 with 1 supplement
Obesity induced Mir802 elevation precedes macrophage accumulation.
(A) mRNA abundance of Mir802 in the epiWAT of Leprdb/db or control mice at 4, 6, 8, 12, and 16 weeks (n=5). (B) mRNA abundance of Mir802 in the epiWAT of mice fed a normal chow diet (NCD) or HFD for 0, 4, 8, 16, 24, and 32 weeks (n=5). (C) The expression level of Mir802 in epiWAT, scWAT and BAT isolated from mice on HFD for 16 weeks or 10 weeks Leprdb/db mice (n=7). (D) Copy number of Mir802 in mature adipocytes and stromal vascular fraction (SVF) of epiWAT isolated from mice on NCD or HFD for 16 weeks (n=5). (E–F) Mir802 expression levels in insulin resistance 3T3-L1 cell models (E) and insulin resistance WAT SVF cells models (F). (G) ChIP assays was performed to test the binding ability between FoxO1 and Mir802 promoter. (H) Mir802 expression levels in the 3T3-L1 cells transfected with ADA-FoxO1 or FoxO1 siRNA. (I) F4/80 and CD11b positive cells in SVFs isolated from the epiWAT of mice fed an HFD for 0, 2, 4, 6, 8, 16, and 24 weeks (n=5). (J) Representative images of F4/80 staining (left) and quantification of crown-like structures (CLSs; middle) and adipose diameter (Right) in the epiWAT of mice fed an HFD (n=5). (K) Expression levels of Mir802 in human subcutaneous adipose tissue (nnormal = 25, nobesity & IR=70). Scatter plots of Mir802 expression versus BMI (L) and HOMA-IR (M). Pearson’s correlation coefficients (r) are shown. The fold of Mir802 was calculated using 2-ΔΔCt. Data represent mean ± SEM. p-values obtained using a two-tailed unpaired Student’s t-test (E, F, K) or two-way ANOVA (A–D, G–J) are indicated. *p<0.05, **p<0.01, ***p<0.001. Relative levels of Mir802 were normalized to U6. epiWAT: epididymal white adipose tissue, scWAT: subcutaneous white adipose tissue, BAT: brown adipose tissue.
Figure 1—figure supplement 1
Obesity induced Mir802 elevation precedes macrophage accumulation.
(A) The mRNA abundance of pri-Mir802 in epiWAT of 4, 6, 8, 12, 16 weeks Leprdb/db mice or control mice (n=5). (B) The mRNA abundance of pri-Mir802 in epiWAT of mice fed with normal chow diet (NCD) or HFD for 0, 4, 8, 16, 24, and 32 weeks (n=5). (C–D) The insulin-resistant cell models were established in 3T3-L1 (C) and WAT SVF cells (D) by 0.5 mM palmitate, 10 μg/ml insulin and 25 mM glucose for 24 hr, and qRT-PCR was performed to measure the expression levels of pri-Mir802. (E) Representative images of flow cytometric analysis of CD11b+/F4/80+ in the SVFs isolated from eipWAT in mice fed with HFD (n=5). (F) The expression levels of pri-Mir802 in the human subcutaneous adipose tissues (nnormal = 25, nobesity & IR=70). (G) FISH analysis of Mir802 in the human subcutaneous adipose tissues of obese patient or normal patient (n=7). The nuclei were stained with DAPI. Magnification:×20, scale bar, 20 μm. Data represent mean ± SEM. The p-values by two-tailed unpaired Student’s t test (C–D, F), or two-way ANOVA (A–B) are indicated. **p<0.01, ***p<0.001. Relative levels of pri-Mir802 were normalized to U6.
Figure 2 with 2 supplements
Adipose tissue-specific overexpression of Mir802 exacerbates adipose tissue inflammation and leads to metabolic dysfunction.
(A) Representative images of F4/80 staining (top), quantification of CLSs (bottom) and adipose diameter (right) in epiWAT of WT or Mir802 KI mice on HFD for 0, 8, and 16 weeks (n=5). Scale bar: 40 μm. (B) Percentage of F4/80+/CD11b+ total macrophages in the epiWAT of Mir802 KI and KI-control mice fed with HFD (n=5). (C) M1 (CD86+CD206–) and M2 (CD206+CD86–) within the macrophage population (n=5). (D) qRT-PCR analysis for mRNA levels of the M1 and M2 markers in the epiWAT of mice on KI-control or Mir802 KI at 16 weeks (n=5). (E–H) Serum levels of CCL2 (E), IL-1β (F), IL-6 (G), and TNF-α (H) of Mir802 KI and control mice fed with HFD for 0, 8, 16, and 30 weeks (n=5). (I, J) Dynamic changes in body weight (I) and glucose (J) in WT and Mir802 KI mice during 30 weeks of HFD feeding (n=5). (K, L) Fat mass of whole body (K) and individual tissues (L) (n=7). (M) Representative coronal section MRI images and visceral and subcutaneous adipose tissue volume of HFD-fed control and Mir802 KI mice (n=5). (N, O) Area over the curve (AOC) of the blood glucose level was calculated via intraperitoneal glucose tolerance tests (IPGTTs, 2 g/kg, N, n=5) or intraperitoneal insulin tolerance tests (IPITTs; 0.75 U/kg, O, n=5). (P) Fasting insulin (FINS) levels of HFD-fed mice were measured by ELISA (n=7). (Q) HOMA-IR was calculated with the equation FBG (mmol l−1)×FINS (mIU l−1)/22.5. Data represent mean ± SEM. Differences between groups were determined by ANOVA (A–J, L, and N–Q) or two-tailed unpaired Student’s t-test (K). *p<0.05, ***p<0.001. Gene levels were normalized to Rn18s abundance.
Figure 2—figure supplement 1
Adipose tissue-specific overexpression of Mir802 exacerbates adipose tissue inflammation.
(A) Schematic diagram showing the strategy for generation of adipose-specific Mir802 KI mice. (B) Genotypic PCR analysis showing that the adipose tissue Mir802 WT mouse carrying homozygous Mir802 KI allele, while KI mouse carrying both KI and Cre allele. (C) qRT-PCR analysis showing a markedly decreased expression of Mir802 in several adipose tissues (epiWAT, scWAT, BAT), but not in liver or heart tissues (n=3). (D) Mir802 mRNA levels in isolated adipocytes and SVF from epiWAT of Mir802 KI mice (n=3). (E) The cumulative food intake of Mir802 KI and control mice treated with NCD feeding (n=5). (F–G) Dynamic changes in body weight (F) and glucose (G) of control and Mir802 KI mice during 30 weeks of NCD feeding (n=7). (H) Fat mass of whole body of control and Mir802 KI mice of NCD feeding (n=7). (I) Representative images of F4/80 in epiWAT of WT or Mir802 KI mice on NCD for 0, 8, and 16 weeks (n=5). Scale bar: 40 μm. (J) Representative images of flow cytometric analysis of CD11b+/F4/80+ cells in the SVFs isolated from eipWAT in control or Mir802 KI mice fed with HFD (n=5). (K) Representative images of flow cytometric analysis of CD86 or CD206 in the SVFs isolate from eipWAT in control or Mir802 KI mice fed with HFD (n=5). Data represent mean ± SEM. Differences between groups were determined by ANOVA (C–G) or two-tailed unpaired Student’s t test (H). ***p<0.001. Mir802 abundance was normalized to U6 level.
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Figure 2—figure supplement 1—source data 1
Related to Figure 2—figure supplement 1B.
The original files of the full raw unedited gels of Mir802 KI mice.
- https://cdn.elifesciences.org/articles/99162/elife-99162-fig2-figsupp1-data1-v2.zip
Figure 2—figure supplement 2
Adipose tissue-specific overexpression of Mir802 leads to metabolic dysfunction.
(A) Representative photos of adipose-specific Mir802 KI mice and their WT Mir802ki/ki littermates fed with either HFD for 16 weeks (n=3). (B–F) IPGTT 1.5 g/kg, (K–O) and IPITT (0.75 U/kg, G–K) were performed in Mir802 KI mice and control mice at the 0th, 4th, 8th, 16th, or 30th week of high-fat diet administration, respectively (n=5). Data represent mean ± SEM. Differences between groups were determined by ANOVA (B–K). ***p<0.001. Mir802 abundance was normalized to U6 level.
Figure 3 with 2 supplements
Adipose tissue–specific ablation of Mir802 protects mice from obesity-induced metabolic dysfunction.
(A–B) Dynamic changes in body weight (A) and glucose (B) of KO control and Mir802 KO mice during 30 weeks of HFD feeding (n=7). (C–D) Fat mass of whole body (C) and individual tissues (D) (n=7). (E) Representative coronal section MRI images and visceral and subcutaneous adipose tissue volume of HFD-fed control and Mir802 KO mice (n=5). (F) Representative images of F4/80 staining (left), quantification of CLSs (middle) and adipose diameter (right) in epiWAT of WT or Mir802 KO mice on HFD for 0, 8, and 16 weeks (n=5). Scale bar: 40 μm. (G) Cells isolated from SVFs of epiWAT in Mir802 KO and WT mice fed with HFD for 8, 16, and 24 weeks were subjected to flow cytometry analysis for percentage of CD11b+/F4/80+ total macrophages (n=5). (H) qRT-PCR analysis for the mRNA levels of the M1 and M2 markers in epiWAT of mice on HFD 16 weeks (n=5). (I) Serum levels of CCL2, IL-1β, IL-6, TNF-α determined with ELISA (n=5). (J–K) AOC of the blood glucose level was calculated via IPGTT (1.5 g/kg, J, n=5) or IPITT (0.75 U/kg, K, n=5). (L) Representative images of F4/80 staining (left), quantification of CLSs (middle) and adipose diameter (right) (n=5) in the epiWAT of WT or Mir802 KO mice. Scale bar: 40 μm. (M–N) The percentage of CD11b+/F4/80+ total macrophages (M, n=5) and M1 (CD86+CD206–), and M2 (CD206+CD86–) within the macrophage population (N, n=5) in the SVFs isolated from epiWAT in the HFD-control or Mir802 eWAT KD/HFD mice. (O) Serum levels of TNF-α, IL-6, IL-1β, CCL2 determined with ELISA (n=6). (P) IPGTT was performed in HFD-control mice or Mir802 eWAT KD/HFD mice(n=5). Data represent mean ± SEM. Differences between groups were determined by ANOVA (A–B, D, E–K, N–P) or two-tailed unpaired Student’s t test (C, L–M). ***p<0.001. Gene levels were normalized to Rn18s abundance.
Figure 3—figure supplement 1
Adipose tissue–specific ablation of Mir802 protects mice from obesity-induced metabolic dysfunction.
(A) Schematic diagram showing the strategy for generation of adipose-specific Mir802 KO mice. (B) Genotypic PCR analysis showing that the adipose tissue Mir802 WT mouse carrying homozygous Mir802 KO allele, while Mir802 KO mouse carries both Mir802 KO and Cre allele. (C) qRT-PCR analysis showing a markedly decreased expression of Mir802 in several adipose tissues (epiWAT, scWAT, BAT), but not in liver or heart tissues (n=3). (D) Mir802 mRNA levels in isolated adipocytes and SVF from epiWAT of Mir802 KO mice (n=3). (E) The cumulative food intake of Mir802 KO and control mice treated with NCD feeding (n=7). (F–G) Dynamic changes in glucose (F) and body weight (G) of control and Mir802 KO mice during 30 weeks of NCD feeding (n=7). (H–I) Fat mass of whole body (H) and individual tissues (I) of control and Mir802 KO mice of NCD feeding (n=7). (J) Representative photos of adipose-specific Mir802 KO mice and their WT Mir802fl/fl littermates fed with either HFD for 16 weeks (n=3). (K) Cells isolated from SVFs of epiWAT in Mir802 KO and KO control mice fed with HFD for 8, 16, and 24 weeks were subjected to flow cytometry analysis for percentage of CD11b+/F4/80+ total macrophages (n=5). (L) Representative images of F4/80 in epiWAT of WT or Mir802 KO mice on NCD for 0, 8, and 16 weeks (n=5). Scale bar: 40 μm. IPGTT (M–O) and IPITT (P–R) were performed in Mir802 KO mice and control mice at the 8th, 16th or 30th week of high-fat diet administration, respectively (n=5). Data represent mean ± SEM. Data represent mean ± SEM. Differences between groups were determined by ANOVA (C–I, M–R). **p<0.01, ***p<0.001. Mir802 abundance was normalized to U6 level.
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Figure 3—figure supplement 1—source data 1
Related to Figure 3—figure supplement 1B.
The original files of the full raw unedited gels of Mir802 KO mice.
- https://cdn.elifesciences.org/articles/99162/elife-99162-fig3-figsupp1-data1-v2.zip
Figure 3—figure supplement 2
Knockdown Mir802 in the eWAT of HFD mice restores adipose function.
(A) Flowchart of the in vivo experiments designed for detecting adipose tissue inflammation and metabolic function via inguinal fat pad infusion of AAV-Adipoq-anti-Mir802 (Mir802 eWAT KD, n=10). (B) The expression levels of Mir802 in the different tissue of HFD-control mice or Mir802 eWAT KD/HFD mice (n=3). (C) Dynamic changes in body weight of Mir802 eWAT KD/HFD mice and control during 8 weeks of HFD feeding. (D) Representative images of flow cytometric analysis of CD11b+/F4/80+ in the SVFs isolated from eipWAT in HFD-control or Mir802 eWAT KD /HFD mice (n=5). (E) Representative images of flow cytometric analysis of CD86 or CD206 in the SVFs isolated from eipWAT in HFD-control or Mir802 eWAT KD /HFD mice (n=5). (F) IPITT was performed in HFD-control mice or Mir802 eWAT KD/HFD mice (n=5). Data represent mean ± SEM. Data represent mean ± SEM. Differences between groups were determined by ANOVA (B–C, F). **p<0.01, ***p<0.001. Mir802 abundance was normalized to U6 level.
Figure 4 with 2 supplements
Interplay between Mir802 ectopically expressed adipocytes and macrophages.
(A) Flowchart of the co-culture experiments designed for determining WAT SVF of obese adipose tissue can affect macrophages (bone marrow derived macrophages BMDMs). (B) Obesity promoted BMDMs migration and invasion in transwell migration and invasion assay. (C) M1 (CD86+CD206–) and M2 (CD206+CD86–) within the macrophage population. (D) The levels of TNF-α, IL-6, IL-1β, and CCL2 determined with ELISA. (E) Migration and invasion ability of BMDMs treated with a medium conditioned with obese or lean SVF cells. (F) The Mir802 expression levels in the BMDMs after co-cultured WAT SVF cells. (G) Chemokine levels in the medium conditioned with obese or lean SVF cells. (H) The Mir802 expression levels in the RAW264.7 cells after co-cultured with 3T3-L1 cells transfected with Mir802 mimics or Mir802 inhibitor. (I) Mir802 induced 3T3-L1 cells recruitment more RAW 264.7 cells in transwell migration and invasion assay. (J) Mir802 mimics-transfected 3T3-L1 cells promoted RAW 264.7 cells M1-like polarization. (K–M) Clodronate-conjugated liposomes (CLOD-liposomes) was injected into WT and Mir802 KI mice. Serum levels of TNF-α, IL-6, IL-1β, CCL2 determined with ELISA n=5, (K); serum insulin levels tested with ELISA n=5, (L); glucose tolerance tested by IPGTT n=7, (M). Data represent mean ± SEM. Differences between groups were determined by ANOVA (D–E, G–M), or two-tailed unpaired Student’s t test (F). **p<0.01, ***p<0.001.
Figure 4—figure supplement 1
Interplay between Mir802 ectopically expressed adipocytes and macrophages.
(A–B) Obesity induced macrophages proliferation tested by EdU staining (A) and flow cytometry analysis FACS, (B). (C) Representative images of flow cytometric analysis of CD86 or CD206 in the SVFs isolated from NCD and HFD mice (n=5). (D) Flowchart of the co-culture experiments designed for determining 3T3-L1cells modified Mir802 expression levels can affect macrophages. (E) qRT-PCR was performed to test the Mir802 expression levels in the 3T3-L1 cells transfected with Mir802 mimics or Mir802 inhibitor. (F–G) EdU staining (F) and FACS analysis (G) were used to detect the proliferation of RAW264.7 cells. (H) FACS analysis of LipidTOX in RAW 264.7 cells. (I) Representative images of flow cytometric analysis of CD86 or CD206 in the 3T3-L1 cells transfected with Mir802 or anti-Mir802. Differences between groups were determined by ANOVA (E, F, G, H). **p<0.01, ***p<0.001. Mir802 abundance was normalized to U6 level.
Figure 4—figure supplement 2
Interplay between Mir802 ectopically expressed adipocytes and macrophages.
(A) The CCL2 levels were determined with ELISA. (B) Mir802 mimics or Mir802 inhibitor were transfected in 3T3-L1 cells, ELISA was performed to test TNF-α, IL-6, IL-1β, and CCL2 levels. (C) Transwell migration and invasion assay were performed to test macrophage migration/invasion behavior in the absence adipocytes or in the of presence of adipocytes and modulation of Mir802 expression. (D) The HOMA-IR of WT and Mir802 KI mice injected with clodronate-conjugated liposomes (CLOD-liposomes). (E) Schematic representation of Mir802’s role in facilitating the recruitment of macrophages into adipose tissue through adipose-secreted CCL2. Data represent mean ± SEM. Differences between groups were determined by ANOVA (A–D). **p<0.01, ***p<0.001. Mir802 abundance was normalized to U6 level.
Figure 5 with 1 supplement
Adipose Mir802 modulates recruitment and polarization of macrophages by directly targeting Traf3.
(A) Heat map illustrating the differential expression of mRNAs in the epiWAT of Mir802 KI mice compared to their WT Mir802ki/ki littermates (n=3). (B) mRNA and protein levels of TRAF3 in human subcutaneous adipose tissues from obese and normal individuals (nnormal = 4 and nobesity&IR=9). (C, D) mRNA and protein levels of TRAF3 in the epiWAT of HFD mice (C, n=3–5) or Mir802 KI mice (D, n=3–5). (E) Relative luciferase activity in 3T3-L1 cells co-transfected with Mir802 mimics and a luciferase reporter containing either Traf3-WT or Traf3-MUT. Data are presented as the relative ratio of Renilla luciferase activity to firefly luciferase activity. (F) Protein levels of TRAF3 in 3T3-L1 cells transfected with Mir802 mimics or Mir802 inhibitor. (G) Anti-Ago2 RIP was performed in 3T3-L1 cells transiently overexpressing Mir802, followed by qRT-PCR to detect Traf3 associated with Ago2 (nonspecific IgG served as a negative control). (H) mRNA and protein levels of TRAF3 in the epiWAT of control, Mir802 KI, Traf3 eWAT OE, and Mir802 KI and Traf3 eWAT OE mice (n=3–5). (I) Representative images of F4/80 staining (left), quantification of CLSs (middle) and adipose diameter (right, n=5). (J) M1 (CD86+CD206–) and M2 (CD206+CD86–) within the macrophage population (n=5). (K) qRT-PCR analysis of the mRNA levels of M1 and M2 markers in the epiWAT of HFD-fed control, Traf3 eWAT OE Mir802 KI, and Mir802 KI & Traf3 eWAT OE(n=5). (L, M) Dynamic changes in body weight (L), glucose level (M), fat mass (N), glucose tolerance (O), and HOMA-IR (P) of control, Mir802 KI, Traf3 eWAT OE and Mir802 KI and Traf3 eWAT OE mice during 30 weeks of HFD feeding (n=7). Data represent mean ± SEM. Differences between groups were determined by ANOVA (E–P). ***p<0.001. Mir802 abundance was normalized to U6 level, and other genes levels were normalized to Rn18s abundance.
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Figure 5—source data 1
Related to Figure 5B.
The original files of the full raw unedited blots of TRAF3 and β-Actin in human subcutaneous adipose tissues from obese and normal individuals (nnormal = 4 and nobesity&IR=9).
- https://cdn.elifesciences.org/articles/99162/elife-99162-fig5-data1-v2.zip
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Figure 5—source data 2
Related to Figure 5C.
The original files of the full raw unedited blots of TRAF3 and β-Actin in the epiWAT of HFD mice (n=3).
- https://cdn.elifesciences.org/articles/99162/elife-99162-fig5-data2-v2.zip
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Figure 5—source data 3
Related to Figure 5D.
The original files of the full raw unedited blots of TRAF3 and β-Actin in the epiWAT of Mir802 KI mice (n=3).
- https://cdn.elifesciences.org/articles/99162/elife-99162-fig5-data3-v2.zip
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Figure 5—source data 4
Related to Figure 5F.
The original files of the full raw unedited blots of TRAF3 and β-Actin in 3T3-L1 cells transfected with Mir802 mimics or Mir802 inhibitor.
- https://cdn.elifesciences.org/articles/99162/elife-99162-fig5-data4-v2.zip
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Figure 5—source data 5
Related to Figure 5H.
The original files of the full raw unedited blots of TRAF3 and β-Actin in the epiWAT of control, Mir802 KI, Traf3 eWAT OE, and Mir802 KI and Traf3 eWAT OE mice (n=3).
- https://cdn.elifesciences.org/articles/99162/elife-99162-fig5-data5-v2.zip
Figure 5—figure supplement 1
Adipose Mir802 modulates recruitment and polarization of macrophages by directly targeting Traf3.
(A) miRPathDB, Targetscanand and multiMiR were used to predict the target genes of Mir802. (B–D) The mRNA and protein levels of TRAF3 in the epiWAT of Lepob/ob mice (B, n=3), Leprdb/db mice (C, n=3) or Mir802 KO mice (D, n=3). (E) mRNA levels of Traf3 in 3T3-L1 cells transfected with Mir802 mimics or Mir802 inhibitor. (F) Anti-Ago2 RIP was performed in 3T3-L1 cells transiently overexpressing Traf3, followed by qRT-PCR to detect Mir802 associated with Ago2 (nonspecific IgG served as a negative control). (G–H) Cells isolated from SVFs of epiWAT in control, Traf3 eWAT OE, Mir802 KI and Mir802 KI & Traf3 eWAT OE, mice fed with HFD 16 weeks were subjected to flow cytometry analysis for percentage of CD11b+/F4/80+ total macrophages (F, n=3) and M1 (CD86+CD206–) and M2 (CD206+CD86–) within the macrophage population (G, n=3). (I) Representative coronal section MRI images and visceral and subcutaneous adipose tissue volume of HFD-fed control, Traf3 eWAT OE, Mir802 KI and Mir802 KI & Traf3 eWAT OE mice. (J) Insulin tolerance test after mice were fed with HFD 16 weeks. (K) Serum insulin levels of control, Mir802 KI, Traf3 eWAT OE and Mir802 KI & Traf3 eWAT OE mice during 30 weeks of NCD or HFD feeding (n=7). Data represent mean ± SEM. Differences between groups were determined by ANOVA (E–F, J–K). ***p<0.001. Mir802 abundance was normalized to U6 level, and other genes levels were normalized to Rn18s abundance.
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Figure 5—figure supplement 1—source data 1
Related to Figure 5—figure supplement 1B.
The original files of the full raw unedited blots of TRAF3 and β-Actin in the epiWAT of Lepob/ob mice (n=3).
- https://cdn.elifesciences.org/articles/99162/elife-99162-fig5-figsupp1-data1-v2.zip
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Figure 5—figure supplement 1—source data 2
Related to Figure 5—figure supplement 1C.
The original files of the full raw unedited blots of TRAF3 and β-Actin in the epiWAT of Leprdb/db mice (n=3).
- https://cdn.elifesciences.org/articles/99162/elife-99162-fig5-figsupp1-data2-v2.zip
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Figure 5—figure supplement 1—source data 3
Related to Figure 5—figure supplement 1D.
The original files of the full raw unedited blots of TRAF3 and β-Actin in the epiWAT of Mir802 KO mice (n=3).
- https://cdn.elifesciences.org/articles/99162/elife-99162-fig5-figsupp1-data3-v2.zip
Figure 6 with 1 supplement
Mir802 activates noncanonical and canonical NF-κB pathways by recruiting macrophages.
(A, B) NIK protein levels in 3T3-L1 cells transfected with Mir802 mimics or Mir802 inhibitor (A) in the epiWAT of Mir802 KI mice (B, n=3). (C, D) P100/52 protein levels in 3T3-L1 cells transfected with Mir802 mimics or Mir802 inhibitor (C), and in the epiWAT of Mir802 KI mice (D, n=3). (E, F) Protein levels of IKK-α and P-IKK-α in 3T3-L1 cells transfected with Mir802 mimics or Mir802 inhibitor (E) in the epiWAT of Mir802 KI mice (F, n=3). (G, H) Overexpression of Traf3 reverses the protein levels of NIK, P-IKK-α, and P100/52 in 3T3-L1 cells (G) and in the epiWAT of Mir802 KI mice (H, n=3). (I, J) Protein levels of some major canonical NF-κB signaling targets in the epiWAT of Mir802 KI mice (I, n=3) and Traf3 eWAT OE rescued mice (J, n=3). (K, L) qRT-PCR (K) and ELISA (L) were performed to detect major chemokine levels. Data represent mean ± SEM. Differences between groups were determined by ANOVA (K–L). ***p<0.001. Genes levels were normalized to Rn18s abundance.
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Figure 6—source data 1
Related to Figure 6A.
The original files of the full raw unedited blots of TRAF3, NIK, and β-Actin in 3T3-L1 cells transfected with Mir802 mimics or Mir802 inhibitor.
- https://cdn.elifesciences.org/articles/99162/elife-99162-fig6-data1-v2.zip
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Figure 6—source data 2
Related to Figure 6B.
The original files of the full raw unedited blots of TRAF3, NIK, and β-Actin in the epiWAT of Mir802 KI mice (n=3).
- https://cdn.elifesciences.org/articles/99162/elife-99162-fig6-data2-v2.zip
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Figure 6—source data 3
Related to Figure 6C.
The original files of the full raw unedited blots of p100/p52 and β-Actin in 3T3-L1 cells transfected with Mir802 mimics or Mir802 inhibitor.
- https://cdn.elifesciences.org/articles/99162/elife-99162-fig6-data3-v2.zip
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Figure 6—source data 4
Related to Figure 6D.
The original files of the full raw unedited blots of p100/p52 and β-Actin in the epiWAT of Mir802 KI mice (n=3).
- https://cdn.elifesciences.org/articles/99162/elife-99162-fig6-data4-v2.zip
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Figure 6—source data 5
Related to Figure 6E.
The original files of the full raw unedited blots of P-IKK-α, IKK-α, and β-Actin in 3T3-L1 cells transfected with Mir802 mimics or Mir802 inhibitor.
- https://cdn.elifesciences.org/articles/99162/elife-99162-fig6-data5-v2.zip
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Figure 6—source data 6
Related to Figure 6F.
The original files of the full raw unedited blots of P-IKK-α, IKK-α, and β-Actin in the epiWAT of Mir802 KI mice (n=3).
- https://cdn.elifesciences.org/articles/99162/elife-99162-fig6-data6-v2.zip
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Figure 6—source data 7
Related to Figure 6G.
The original files of the full raw unedited blots of p100/p52, P-IKK-α, IKK-α, NIK, and β-Actin in the 3T3-L1 cells.
- https://cdn.elifesciences.org/articles/99162/elife-99162-fig6-data7-v2.zip
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Figure 6—source data 8
Related to Figure 6H.
The original files of the full raw unedited blots of p100/p52, P-IKK-α, IKK-α, NIK, and β-Actin in the epiWAT of Mir802 KI and Traf3 eWAT OE mice (n=3).
- https://cdn.elifesciences.org/articles/99162/elife-99162-fig6-data8-v2.zip
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Figure 6—source data 9
Related to Figure 6I.
The original files of the full raw unedited blots of some major canonical NF-κB signaling targets in the epiWAT of Mir802 KI mice(n=3).
- https://cdn.elifesciences.org/articles/99162/elife-99162-fig6-data9-v2.zip
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Figure 6—source data 10
Related to Figure 6J.
The original files of the full raw unedited blots of some major canonical NF-κB signaling targets in the epiWAT of Mir802 KI mice and Traf3 eWAT OE rescued mice (n=3).
- https://cdn.elifesciences.org/articles/99162/elife-99162-fig6-data10-v2.zip
Figure 6—figure supplement 1
Mir802 activates noncanonical and canonical NF-κB pathways by recruiting macrophages.
(A) GO analysis of RNA sequencing in epiWAT of Mir802 KI mice compared to their WT Mir802fl/fl littermates. (B) NIK protein levels in the epiWAT of Mir802 KO mice (n=3). (C) P100/52 protein levels in the epiWAT of Mir802 KO mice (n=3). (D) The protein levels of IKK-α and P-IKK-α in the epiWAT of Mir802 KO mice (n=3). Data represent mean ± SEM.
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Figure 6—figure supplement 1—source data 1
Related to Figure 6—figure supplement 1B.
The original files of the full raw unedited blots of TRAF3, NIK, and β-Actin in in the epiWAT of Mir802 KO mice (n=3).
- https://cdn.elifesciences.org/articles/99162/elife-99162-fig6-figsupp1-data1-v2.zip
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Figure 6—figure supplement 1—source data 2
Related to Figure 6—figure supplement 1C.
The original files of the full raw unedited blots of p100/p52 and β-Actin in the epiWAT of Mir802 KO mice (n=3).
- https://cdn.elifesciences.org/articles/99162/elife-99162-fig6-figsupp1-data2-v2.zip
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Figure 6—figure supplement 1—source data 3
Related to Figure 6—figure supplement 1D.
The original files of the full raw unedited blots of P-IKK-α, IKK-α, and β-Actin in the epiWAT of Mir802 KO mice (n=3).
- https://cdn.elifesciences.org/articles/99162/elife-99162-fig6-figsupp1-data3-v2.zip
Figure 7 with 1 supplement
Mir802 promotes lipogenesis and induces M1 macrophage polarization in adipose tissue through activating SREBP1.
(A) Heat map illustrating the top 20 upregulated mRNAs in the epiWAT of Mir802 KI mice compared to their WT Mir802fl/fl littermates (n=3). (B–C) Protein levels of the mature form of SREBP-1 protein (m-SREBP1) and the precursor form of SREBP-1 (P-SREBP1) in mature 3T3-L1 cells transfected with Mir802 mimics or Mir802 inhibitor (B), in the epiWAT of Mir802 KI mice (C, n=3). (D) The protein levels of m-SREBP1 and P-SREBP1 were reversed by Traf3. (E) Srebp1a mRNA levels in 3T3-L1 cells transfected with Rela-overexpressing plasmid or Rela shRNA plasmid. (F, G) ChIP-qPCR assays were conducted to verify that Rela binds to the Srebp1 promoter in 3T3-L1 cells transfected with Mir802 mimics or Mir802 inhibitor (F) and in the epiWAT of Mir802 KI mice (G, n=3). (H) DNA pull-down assay using a biotinylated DNA probe corresponding to the −360 to −400 or −1198 to −1237 region of the Srebp1 promoter in 3T3-L1 cells transfected with Mir802 mimics or Mir802 inhibitor. (I) DNA pull-down assay using a biotinylated DNA probe corresponding to the −1198 to −1237 region of the wild-type (WT) or a mutant sequence of the Srebp1 promoter in 3T3-L1 cells stimulated with Rela plasmid for 48 hr. (J) Luciferase reporter assays in 3T3-L1 cells transfected with the indicated plasmids for 48 hr. Dual-luciferase activity was determined. (K) Mir802 mimics or Mir802 inhibitor was transfected into 3T3-L1 cells, then direct contact co-culture with mature 3T3-L1 and RAW264.7 cells, flow cytometry analysis of cellular neutral lipid content using the LipidTOX in cells. (L) Representative images of the immunofluorescence of lipid droplets (HCS LipidTOX, Red) and F4/80 (Green, n=3). Scale bar: 20 μm. (M) Transmission electron microscopy (TEM) was performed to detect the contact between lipid droplets and macrophages (n=3). Data represent mean ± SEM. Differences between groups were determined by ANOVA (E–G and J). ***p<0.001. Genes levels were normalized to Rn18s abundance.
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Figure 7—source data 1
Related to Figure 7B.
The original files of the full raw unedited blots of m-SREBP1, P-SREBP1, and β-Actin in mature 3T3-L1 cells transfected with Mir802 mimics or Mir802 inhibitor.
- https://cdn.elifesciences.org/articles/99162/elife-99162-fig7-data1-v2.zip
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Figure 7—source data 2
Related to Figure 7C.
The original files of the full raw unedited blots of m-SREBP1, P-SREBP1, and β-Actin in in the epiWAT of Mir802 KI mice (n=3).
- https://cdn.elifesciences.org/articles/99162/elife-99162-fig7-data2-v2.zip
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Figure 7—source data 3
Related to Figure 7D.
The original files of the full raw unedited blots of m-SREBP1, P-SREBP1, and β-Actin in the 3T3-L1 cells.
- https://cdn.elifesciences.org/articles/99162/elife-99162-fig7-data3-v2.zip
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Figure 7—source data 4
Related to Figure 7H.
The original files of the full raw unedited blots of p65 and β-Actin in 3T3-L1 cells transfected with Mir802 mimics or Mir802 inhibitor.
- https://cdn.elifesciences.org/articles/99162/elife-99162-fig7-data4-v2.zip
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Figure 7—source data 5
Related to Figure 7I.
The original files of the full raw unedited blots of p65 and β-Actin in 3T3-L1 cells stimulated with Rela plasmid for 48 hr.
- https://cdn.elifesciences.org/articles/99162/elife-99162-fig7-data5-v2.zip
Figure 7—figure supplement 1
Mir802 promotes lipogenesis and induces M1 macrophage polarization in adipose tissue through activating SREBP1.
(A–B) qRT-PCR was performed to detect Srebp1a mRNA levels in the 3T3-L1 cells Mir802 mimics or Mir802 inhibitor (A) and in the epiWAT of Mir802 KI mice (B, n=3). (C) The predicted binding site of Rela on the Srebp1 promoter. (D–E) ChIP-PCR experiments were conducted to verify that Rela binds to the promoter of Srebp1 in the 3T3-L1 cells (D) and in the epiWAT of Mir802 KI mice (E, n=3). (F) Schematic illustration for the mechanism of Mir802 increased Srebp1 expression by activating canonical NF-κB pathways. (G) Representative images of immunofluorescence of lipid drop (HCS LipidTOXTM, Red) and DAPI (Blue). Scale bar: 20 μm. (H) Oil red O staining was performed to test the lipid droplet number in the 3T3-L1 cells transfected with Mir802 mimics or Mir802 inhibitor. (I) Immunohistochemical analysis was performed to test F4/80, CD86, and CD206 levels in the epiWAT of Mir802 KI mice (n=3), Scale bar: 20 μm. Data represent mean ± SEM. Differences between groups were determined by ANOVA (A–B). ***p<0.001. Genes levels were normalized to Rn18s abundance.
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Figure 7—figure supplement 1—source data 1
Related to Figure 7—figure supplement 1D.
The original files of the full raw unedited gels by ChIP-PCR experiments in the 3T3-L1 cells.
- https://cdn.elifesciences.org/articles/99162/elife-99162-fig7-figsupp1-data1-v2.zip
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Figure 7—figure supplement 1—source data 2
Related to Figure 7—figure supplement 1E.
The original files of the full raw unedited gels by ChIP-PCR experiments in the epiWAT of Mir802 KI mice (n=3).
- https://cdn.elifesciences.org/articles/99162/elife-99162-fig7-figsupp1-data2-v2.zip
Figure 8
Schematic illustration for the mechanism of Mir802 exacerbates adipose tissue inflammation and leads to metabolic dysfunction during obesity.
We found that Mir802 endows adipose tissue with the ability to interact with macrophages and regulate the inflammatory cascade. During obesity, Mir802 promotes adipose tissue secretion more chemokines to recruit macrophages by targeting Traf3 activating canonical and noncanonical NF-κB signaling pathways; and Mir802 increases lipogenesis through promoting Srebp1 transcription, leading macrophages toward proinflammatory M1 phenotype by engulfing lipid droplets. Schematic illustration was drawn by figdraw.
Additional files
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Supplementary file 1
The supplementary tables in the manuscript.
(a) Clinical characteristics of the patients with obese patients and normal individuals. (b) RNA islolated from epiWAT of wide type mice and Mir802 KI mice, this table shows significantly changed mRNA (Log2 (FPKM (Mir802 KI/WT))≥1). (c) Primer sequences used for RT-PCR. (d) Oligo sequences used for shRNA. € The primers used in Real-time PCR (5’–3’).
- https://cdn.elifesciences.org/articles/99162/elife-99162-supp1-v2.docx
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MDAR checklist
- https://cdn.elifesciences.org/articles/99162/elife-99162-mdarchecklist1-v2.docx
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Source data 1
Raw data of histograms.
- https://cdn.elifesciences.org/articles/99162/elife-99162-data1-v2.xlsx
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Source data 2
Quantification of the western blot analysis.
- https://cdn.elifesciences.org/articles/99162/elife-99162-data2-v2.xls
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Adipocyte microRNA-802 promotes adipose tissue inflammation and insulin resistance by modulating macrophages in obesity
eLife 13:e99162.
https://doi.org/10.7554/eLife.99162
