Fig 2: miR-185-5p directly regulates G6Pase expression. A-B: mRNA (A) and protein (B) expression of gluconeogenic genes (Ppargc1a, PEPCK, and G6Pase) in the livers of C57BL/6J mice administered with miR-185-5p LNA or negative control. The quantification plot was based on scanning densitometry analysis using the ImageJ software (v 1.8.0). C: Sequence alignment of miR-185-5p with the 3'-UTR of the mouse and human G6Pase. D-E: HEK293T cells were co-transfected with wildtype or mutant 3'-UTR reporter plasmids of G6Pase with miR-185-5p mimics (D), miR-185-5p antisense (E). F-G: MPHs were transfected with miR-185-5p antisense or negative control for 48 h and then treated with FSK (10 µM) and DEX (100 nm) for an additional 6 h. Then, mRNA levels of miR-185-5p and gluconeogenic genes (Ppargc1a, PEPCK, and G6Pase) were examined (F) and G6Pase protein level was determined (G); the quantification plot was based on scanning densitometry analysis using the ImageJ software (v 1.8.0). H-I: MPHs were transfected with miR-185-5p antisense or negative control for 48 h and then treated with FSK (10 µM) and DEX (100 nm) for an additional 6 h. Then, cellular glucose production (H) and G6Pase mRNA levels (I) were determined. J-K: MPHs were infected with Ad-miR-185-5p or negative control for 48 h and then treated with FSK (10 µM) and DEX (100 nm) for additional 6 hours. Then, mRNA (J) and protein (K) levels of gluconeogenesis (PGC-1a, PEPCK, and G6Pase) were examined. L-M: MPHs were infected with Ad-miR-185-5p or negative control for 48 h and then treated with FSK (10 µM) and DEX (100 nm) for an additional 6 h. Then, cellular glucose production and G6Pase mRNA levels (L) and G6Pase mRNA levels (M) were determined. **P < 0.01. ***P < 0.001.

Fig 3: Hesperetin induces mitochondrial biogenesis in vitro. (a–h) Primary differentiated myotubes were treated 48 h with hesperetin or DMSO. (a) Custom-made microfluidic gene expression array with mitochondrial and myogenic genes (n = 2). Hesperetin-treated primary differentiated myotubes are marked with H, DMSO control with D. (b) Taqman verification of mitochondrial target genes (n = 4). (c) Taqman verification of Pgc-1alpha gene expression (Ppargc1a) after 48 and 72 h hesperetin stimulation. (d–h) Western Blot analysis and quantification of PGC-1alpha (d,e) and respiratory complexes (f–h). GAPDH served as loading control and was detected with another fluorescent IRDye (representative blots of n = 3 independent experiments). Shown are cropped gels. Tim23 was used to normalize protein levels to mitochondrial content (e,h). (f) Due to higher protein levels of complex V and III, exposure times were reduced for visualization compared to lower expressed respiratory complexes (I, II and IV). Combined blots are shown in Suppl. Fig. S1. Values represent means ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 (two-tailed t-test).

Fig 4: BAT-Specific Ablation of FGF21 Significantly Reduces the Increased BAT Thermogenic Activity, BAT Mass, and Beige Cell Emergence in iWAT Observed from BAT-Specific miR-32 Overexpression(A) FGF21 mRNA expression in BAT but not in liver or iWAT was ablated in miR-32-AAV-BS+Cre mice (n = 6) and control-AAV-BS+Cre mice (n = 6).(B) FGF21 protein expression in BAT but not in liver or iWAT was ablated in miR-32-AAV-BS+Cre mice (n = 6) and control-AAV-BS+Cre mice (n = 6).(C) Quantification of relative protein expression using ImageJ showed that protein level of FGF21 in BAT but not in liver or iWAT was ablated in miR-32-AAV-BS+Cre mice (n = 6) and control-AAV-BS+Cre mice (n = 6).(D) Serum FGF21 levels were decreased in miR-32-AAV-BS+Cre mice (n = 6) and control-AAV-BS+Cre mice (n = 6) compared to wild-type mice (n = 4).(E) miR-32-AAV-BS+Cre mice (n = 6) showed higher core body temperatures only during first 48 hr of cold exposure when compared to control-AAV-BS+Cre mice (n = 6).(F) Total energy expenditure was similar after 7 days’ cold stress in miR-32-AAV-BS+Cre mice (n = 6) as compared with control-AAV-BS+Cre mice (n = 6). Energy expenditure was normalized to lean body mass.(G) Average total energy expenditure was slightly higher in miR-32-AAV-BS+Cre mice (n = 6) than control-AAV-BS+Cre mice (n = 6) but not statistically significant.(H) Percentage BAT mass was slightly higher in miR-32-AAV-BS+Cre mice (n = 6) compared to control-AAV-BS+Cre mice (n = 6).(I) In BAT, mRNA levels of Tob1 were lower in miR-32-AAV-BS+Cre mice (n = 6) compared to control-AAV-BS+Cre mice (n = 6), whereas expression of several thermogenic genes including UCP1 was higher in miR-32-AAV-BS+Cre mice.(J) Protein levels of PGC1a and UCP1 were higher in miR-32-AAV-BS+Cre mice (n = 6) compared to control-AAV-BS+Cre mice (n = 6).(K) Quantification of relative protein expression using ImageJ showed that protein levels of PGC1 and UCP1 were higher in miR-32-AAV-BS+Cre mice (n = 6) compared to control-AAV-BS+Cre mice (n = 6). Average intensities were normalized to that of Calnexin.(L) mRNA levels of thermogenic genes in iWAT were similar in both groups of mice (both n = 6). Data were normalized to PPIA.(M) Immunoblots showed that miR-32-AAV-BS+Cre mice (n = 6) had similar UCP1 and PGC1a protein levels in iWAT compared to control-AAV-BS+Cre mice (n = 6).(N) Quantification of relative UCP1 and PGC1a protein levels using ImageJ. Average intensities were normalized to that of Calnexin.(O) Proposed mechanism by which miR-32 promotes BAT thermogenesis by inhibiting Tob1, activating p38/MAPK signaling and driving UCP1, PGC1a, and FGF21 expression in BAT. The BAT secreted FGF21 functions in a paracrine fashion to promote further thermogenic gene expression in BAT as well as in an endocrine fashion to promote iWAT browning.Data represent mean ± SEM. *p < 0.05, **p < 0.01, and ***p < 0.001. See also Figure S7.

Fig 5: Characterization of TrxR2-Tg mouse.a Breeding schematic of CAG-LoxP-STOP-LoxP-TrxR2 transgenic mice that were bred with EIIA-CRE mice producing CAG-LoxP-TrxR2 mice (referred to as TrxR2-Tg). b, c TrxR2 is elevated in TrxR2-Tg mice. Immunoblots and quantifications of TrxR2 in the brain, muscle, and heart tissues from 10-month-old TrxR2-Tg mice and littermate controls (n = 3). d TrxR2 overexpression does not alter mitochondrial abundance. Immunoblot of TrxR2, VDAC, and PGC-1 in MEFs derived from TrxR2-Tg mice (n = 3). e TrxR2 overexpression is localized in mitochondria. Immunofluoresence of TrxR2-Tg and non-transgenic MEFs stained with mitotracker, representative images. f Western blot of oxidized and total levels PRX protein in TrxR2-Tg versus control liver mitochondria isolates (n = 3). g TrxR2 overexpression increases oxidative stress resistance. H2O2 cell survival assay of TrxR2-Tg and Control MEFS (n = 3). h Tert-butyl hydroperoxide cell survival assay of TrxR2-TG MEFs and non-transgenic MEFS (n = 3). Values are Mean ± SEM. *p < 0.05, **p < 0.01.