MicroRNA-33 Inhibits Adaptive Thermogenesis and Adipose Tissue Beiging

Abstract

[Figure: see text].

Keywords: adipose tissue; autophagy; cholesterol; microRNAs; thermogenesis.

Figure 1.. miR-33 modulates brown adipocyte functionality in vitro.

A-B) Relative expression of (A) mmu-miR-33, mmu-206 and Srebf2, and (B) Prdm16, Ucp1 and Ppargc1a in 10T1/2 cells during brown-like differentiation, as measured by quantitative real-time PCR and normalized to RNU6 (A) or Hprt (B). C-D) Relative expression of (C) mmu-miR-33 and (D) Ucp1 and Ppargc1a in brown-like differentiated 10T1/2 cells treated with either vehicle, 10 μM forskolin for 4h or 20 μM isoproterenol for 1h. E) Representative images of brown-like differentiated 10T1/2 cells transfected with control anti-miR or anti-miR-33 oligonucleotides and stained with BODIPY neutral lipid stain. Lipid droplet quantification shown at right. F-G) Oxygen consumption rate (OCR) of PIBA cells differentiated using a BAT induction cocktail and transfected with control (black) or miR-33 (red) mimics (red). OCR was measured (F) in the basal state or after treatment with oligomycin, FCCP and rotenone + antimycin (RA), and (G) basal, maximal and spare respiratory capacity (SRC) were calculated. H-I) OCR (H) and basal, maximal and SRC (I) of differentiated PIBA cells transfected with control anti-miR (black) or anti-miR-33 (blue) and treated as in (F-G). Data in A-E, G and I are the mean ± SEM of three independent experiments. P-values were calculated by One-Way ANOVA with a Bonferroni’s multiple comparisons test (all time points compared to time 0) (A-B) or two-tailed Student’s t-test (C-I). Data shown in F and H are representative curves from three independent experiments.

Figure 2.. miR-33 targets regulators of thermogenesis.

A) Relative expression of Ppargc1a mRNA in brown-fat differentiated 10T1/2 cells transfected with control mimic, miR-33 mimic, control anti-miR or anti-miR-33, as measured by qPCR and normalized to Hprt. B) Activity of a UCP1 promoter-luciferase reporter gene in differentiated 10T1/2 cells transfected with control anti-miR or anti-miR-33 in the presence of control or Ppargc1a-targeting shRNAs. C) Predicted miR-33 target genes involved in adaptive thermogenesis. D) Putative miR-33 binding sites in the 3’-UTRs of Prdm16, Zfp516 and Dio2. E-F) Relative luciferase activity in HEK293T cells transfected with reporter constructs containing the 3’-UTRs of Dio2, Zfp516 and Prdm16, co-transfected with (E) control mimic or miR-33 mimic or (F) control anti-miR or anti-miR-33. G) Relative expression of Dio2, Zfp516 and Prdm16 mRNA in PIBA cells transfected with control mimic, miR-33 mimic, control anti-miR or anti-miR-33, as measured by qPCR and normalized to Hprt. Data in A, B, E-G are the mean ± SEM of three independent experiments. P values were calculated by two-tailed Student’s t-test.

Figure 3.. miR-33 antagonism promotes thermogenesis.

A) Relative expression levels of miR-33 in brown adipose tissue (BAT) and epididymal white adipose tissue (WAT) from C57BL6 mice housed at room temperature (RT) (n=4). B-D) Relative expression levels of (B) miRNAs normalized to RNU6, (C) miR-33 target genes, and (D) Ucp1 mRNA in BAT of C57BL6/J mice kept at RT or 4°C for 6 hours (n=4–5/group). E) Experimental outline of cold challenge in control anti-miR and anti-miR-33 treated C57BL6/J mice. F) Representative immunofluorescence staining for the 2′F/MOE-modified control anti-miR or anti-miR-33 oligonucleotides in BAT. Scale bar = 10 μm. G-H) Core body temperature of control anti-miR and anti-miR-33 treated mice before (G) and during cold exposure (H) (n=5/group). Data are the mean ± SEM. P values were calculated by Student’s t test (A, B, C ,D, G) or two-way ANOVA with a Bonferroni’s multiple comparisons test (H).

Figure 4.. In vivo inhibition of miR-33 upregulates UCP-1 levels in BAT and reduces lipid stores.

A) Relative mRNA expression levels of miR-33 target genes and Ucp1 in BAT from C57BL6/J mice treated with control anti-miR or anti-miR-33 and exposed to cold for 4 hours (n=3 mice/group). B-C) Western blot analysis (B) and quantification (C) of miR-33 target genes and Ucp1 protein levels in BAT from C57BL6/J mice treated with control anti-miR or anti-miR-33 and exposed to cold for 4 hours (n=3 mice/group). D) Immunofluorescence staining for UCP-1 in BAT of C57BL6/J mice treated with control anti-miR or anti-miR-33 and exposed to cold for 4 hours. Quantification shown at right (n=4–5 mice/group). Scale bar 50 μm. E) Representative images of hematoxylin and eosin stained BAT and quantification of adipocyte size in C57BL6/J mice treated with control anti-miR or anti-miR-33 and exposed to cold for 4 hours (n=5 mice/group). Scale bar 50 μm. F) Plasma levels of triglycerides and free fatty acids (FFA) in C57BL6/J mice treated with control anti-miR or anti-miR-33 and exposed to cold for 4 hours (n=4 ctrl anti-miR and 5 anti-miR33). Data are the mean ± SEM. P values were calculated by Student’s t test (C-F).

Figure 5.. In vivo inhibition of miR-33 promotes beige cell emergence in subcutaneous iWAT.

A) Weights of brown (BAT), inguinal (iWAT) and epididymal (eWAT) adipose tissue depots in C57BL6/J mice treated with control anti-miR or anti-miR-33 and exposed to cold for 4 hours. B) Immunofluorescence staining for UCP1 in iWAT of C57BL6/J mice treated with control anti-miR or anti-miR-33 and exposed to cold for 4 hours. Quantification shown at right. (n=4 mice/group). Scale bar 25 μm. C) Relative mRNA expression levels of miR-33 target genes and Ucp1 in iWAT of C57BL6/J mice treated with control anti-miR or anti-miR-33 and exposed to cold for 4 hours (n=5 mice/group). D) Representative images of hematoxylin and eosin stained iWAT and quantification of adipocyte size in C57BL6/J mice treated with control anti-miR or anti-miR-33 and exposed to cold for 4 hours (n=5 mice/group). Scale bar 100 μm. E) Ratio of plasma adiponectin to leptin in C57BL6/J mice treated with control anti-miR or anti-miR-33 and exposed to cold for 4 hours (n=5 mice/group). Data are the mean ± SEM. P values were calculated by Student’s t-test (A -E).