The brown fat-enriched exosomal miR-206-3p attenuates hepatic lipogenesis by decreasing pentose phosphate pathway

Figures

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  Figure 1. Characterization and the miRNA profile of BAT-derived exosomes from WT and obese mice. (a) Schematic diagram of exosome isolation from adipose tissue. (b) Representative electron micrographs of exosomes isolated from BAT. Scale bar, 100 nm. (c) NTA of exosome size distribution and particle concentration (n = 3). (d) Bicinchoninic acid (BCA) analysis of exosome protein concentration (n = 3). (e) Western blot detection of exosomal markers ALIX, TSG101, and the fatty acid-binding protein (FABP4) (iWAT mass: 0.34 g/mouse; gWAT mass: 0.39 g/mouse; BAT mass: 0.11 g/mouse). Exosomes were isolated from equal volumes of culture medium derived from iWAT, gWAT, and BAT of 8-week-old male C57BL/6J mice (c−e). (f) NTA of exosome size distribution and particle concentration (n = 3). (g) BCA analysis of exosome protein concentration (n = 3). (h) Western blot detection of exosomal markers ALIX, TSG101, CD9, and FABP4 (WT BAT mass: 0.12 g/mouse; ob/ob BAT mass: 0.38 g/mouse). Exosomes were isolated from equal volumes of BAT culture medium from 8-week-old WT and ob/ob mice (f−h). (i) Heatmap of the miRNA sequencing comparing BAT-derived exosomes from WT and ob/ob mice (n = 4). (j) RT-qPCR…

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  Figure 2. BAT-derived miR-206-3p is downregulated by obesity and upregulated by exercise. (a) RT-qPCR analysis of relative miR-206-3p levels in iWAT, gWAT, BAT, liver, and muscle from male C57BL/6J mice at 8 weeks of age (n = 6). (b) FPKM value of miR-206-3p levels in iWAT, gWAT, and BAT-derived exosomal miRNA sequencing from male C57BL/6J mice at 8 weeks of age (n = 4). (c) RT-qPCR analysis of the relative levels of miR-206-3p and Ucp1 in SVF and mature adipocytes (ADI) obtained from BAT of male C57BL/6J mice at 8 weeks of age (n = 6). (d) Representative images of H&E staining and miRNAscope staining for miR-206-3p, and immunohistochemical staining for UCP1 in BAT from male C57BL/6J mice at 8 weeks of age. Scale bars, 2.5 mm (up) and 50 μm (BAT). (e) Representative images of H&E staining and miRNAscope staining for miR-206-3p in muscle obtained from male C57BL/6J mice at 8 weeks of age. Scale bars, 2.5 mm (up) and 10 μm (Muscle). (f) Representative images of H&E staining and miRNAscope staining for miR-206-3p in BAT obtained from male C57BL/6J mice fed ND or HFD for 16 weeks starting at the age of 8 weeks. Scale bars, 50 μm (partial) and 2.5 mm (whole). (g−i) RT-qPCR analysis performed to…

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  Figure 3. BAT exosome-mediated hepatic delivery of miR-206-3p. (a) C57BL/6J mice injected with PBS or 50 μg PKH26-labeled exosomes derived from iWAT, gWAT, or BAT and subjected to IVIS analysis at 24 h after intravenous (i.v.) injection. (b) Representative images of primary hepatocytes transfected with Cy3-labeled NC mimic or miR-206-3p mimic for 24 h. Scale bar, 20 μm. (c) Representative images of primary hepatocytes co-cultured with brown adipocytes-derived miR-206-3p-overexpressing exosomes. Brown adipocytes were transfected with miR-206-3p mimic or NC mimic. The exosomes secreted by adipocytes were labeled with PKH26 and co-cultured with primary hepatocytes for 24 h. Scale bar, 50 μm. (d) RT-qPCR analysis of relative miR-206-3p levels in brown adipocytes derived exosomes. Primary brown adipocytes were transfected with miR-206-3p mimic or NC mimic for 24 h, followed by induction of cell differentiation and collection of culture supernatants for exosome isolation (n = 3). (e) RT-qPCR analysis of relative miR-206-3p levels in primary hepatocytes pretreated with the conditioned media from mature adipocytes transfected with NC mimic or miR-206-3p mimic. Mature adipocytes were seeded in the…

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  Figure 4. Adipose-specific miR-206 deletion exacerbates obesity-associated metabolic dysfunction in mice. (a) Body weight of WT and AKO male mice after HFD (n = 8). (b) The iWAT, gWAT, BAT, and liver weight in the indicated male mice (n = 7−9). (c) Body weight of WT and AKO female mice after HFD (n = 8). (d) The iWAT, gWAT, BAT, and liver weight in the indicated female mice (n = 8). (e) Representative images of H&E and UCP1 staining in BAT from WT and AKO male mice. Scale bars, 100 μm. (f) Relative mRNA expression of adipogenesis (aP2) and thermogenic (Ucp1 and Pgc1α) genes in BAT (n = 6). (g) Glucose concentrations during the GTT in male mice after 11 weeks of HFD feeding (n = 6). (h) Glucose concentrations during the ITT in male mice after 10 weeks of HFD feeding (n = 6). (i) Glucose concentrations during the GTT in female mice after 11 weeks of HFD feeding (n = 6). (j) Glucose concentrations during the ITT in female mice after 10 weeks of HFD feeding (n = 6). (k) Oxygen consumption rate (VO2) measured by metabolic cages for male mice after 16 weeks of HFD feeding (n = 12). (l) Carbon dioxide consumption rate (VCO2) measured by metabolic cages for male mice after 16 weeks of HFD feeding…

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  Figure 5. miR-206-3p deficiency exacerbates hepatic steatosis. (a) Gross morphology of livers from 24-week-old WT and AKO male mice fasted for 16 h and H&E staining of liver sections from these mice (n = 7−9). Scale bar, 100 μm. (b) Liver TG levels in male mice (n = 7−9). (c) The levels of serum TG, TC, LDL, HDL, AST, and ALT in male mice (n = 6). (d) Gross morphology of livers from 24-week-old WT and AKO female mice fasted for 16 h and H&E staining of liver sections from these mice (n = 7−9). Scale bar, 100 μm. (e) Liver TG levels in female mice (n = 6). (f) The levels of serum TG, TC, LDL, HDL, AST, and ALT in female mice (n = 6). The 8-week-old WT and AKO mice were fed HFD for 16 weeks before being sacrificed for analysis (a−f). (g) Representative images of BODIPY (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene) and DAPI fluorescence in primary hepatocytes. Scale bar, 20 μm. (h) TG levels in primary hepatocytes (n = 3). Brown adipocytes were transfected with miR-206-3p mimic or NC mimic for 24 h. Following differentiation induction, culture supernatants were harvested and co-incubated with primary hepatocytes for 24 h (g and h). (i−n) Impact of miR-206-overexpressing adenovirus injection…

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  Figure 6. miR-206-3p attenuates lipogenesis by targeting the PPP. (a) Heatmap of liver metabolomics analysis. The 8-week-old male C57BL/6J mice were fed HFD for 16 weeks and injected via tail vein with Ad-GFP or Ad-miR-206 once weekly beginning at week 13 (n = 4). (b) Metabolomics identified up-regulated (red) and down-regulated (blue) metabolites in Ad-miR-206-injected mice. (c) NADPH/NADP+ ratios in primary hepatocytes transfected with NC mimic or miR-206 mimic (n = 3). (d) Relative levels of acetyl-CoA and glycerol-3-phosphate identified by metabolomics (n = 4). (e) RT-qPCR analysis of relative miR-206-3p levels in primary hepatocytes (n = 3). (f) Relative mRNA expression of PPP genes (G6pd, Pgd, and Tkt) in primary hepatocytes (n = 3). (g) Western blot analysis of PPP proteins (G6PD, PGD, and TKT) in primary hepatocytes. Primary hepatocytes were transfected with NC mimic, miR-206 mimic, or miR-206-3p mimic plus miR-206-3p inhibitor. After 48 h, cells were harvested for RNA and protein analyses (e−g). (h) Relative mRNA expression of PPP genes (G6pd, Pgd, and Tkt) analyzed in hepatocytes incubated with conditioned medium from mature adipocytes (n = 3). Brown adipocytes were transfected…

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  Figure 7. miR-206-3p is reduced in human obesity. (a) qPCR analysis of relative miR-206-3p levels in serum-derived exosomes from healthy controls (Non-MAFLD, n = 24) and MAFLD patients (n = 32). (b−g) Correlations between (b) BMI, (c) waist, (d) TG, (e) TC, (f) LDL, and (g) APOB and the expression levels of miR-206-3p in serum-derived exosomes (n = 50). (h) Flowchart of co-culture between human SVF and hepatocytes. (i) Relative Pparγ and miR-206-3p expression in human mature adipocytes (n = 4). (j) Relative mRNA expression of PPP genes (G6pd, Pgd, and Tkt) in hepatocytes incubated with conditioned medium from mature adipocytes (n = 4). Human-derived SVF was induced to differentiate into mature adipocytes, and the culture medium was harvested on day 8 after induction for further study (h−j).(k) Representative H&E, G6PD, PGD, and TKT staining in liver sections of MAFLD patients and Non-MAFLD controls. Scale bar, 100 μm. (l) Quantification of positive area for immunohistochemistry staining in the liver (n = 6). Values are means ± SD. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 by Student’s t test or ANOVA test.