Fig 2: Expression of specific genes involved in energy expenditure is reduced in MTKO mice and increased in UBX mice.a–e, RNA was prepared from quadriceps muscles of ad lib fed 10-week old WT, MTKO, and UBX mice, and qPCR was used to measure the relative abundances of selected transcripts. N=4 WT, 5 MTKO, and 4 UBX mice except for N=3 for UBX TUG-Cter data (a). a, Control reactions were used to verify knockout of TUG in MTKO mice and expression of the UBX-Cter transgene in UBX mice. b, Relative abundances of transcripts for Sarcolipin (Sln), Ucp1, and β3-adrenergic receptor (Adrb3) are shown. c, Relative abundances of the indicated transcripts involved in energy expenditure and lipid metabolism are shown. d, Relative abundances of transcripts encoding PGC-1α (Ppargc1a) and PPARγ (Pparg) are shown. e, Relative abundances of transcripts encoding the indicated calsequestrin proteins are shown. Data are presented as mean ± SEM of biologically independent samples, analyzed using two-tailed t-tests (a–e). f, Gene set enrichment analysis was done on transcripts that were differentially expressed in quadriceps of 11-week old, 4–6 h fasted UBX mice, compared to WT controls (N=3 mice in each group; transcripts were analyzed using CuffDiff as described in the Methods section). Transcripts were ranked in order of significance, and the top 2000 transcripts were analyzed using the GO Biological Process ontology gene set. The gene set corresponding to “Temperature Homeostasis” was significantly enriched (Fig. 3b; False Discovery Rate q value = 0.038)). Here, specific genes in this set that were differentially expressed in UBX vs. WT muscles are listed, together with their rank in the gene list, the fold-change in expression in UBX muscles compared to controls, the adjusted p-value in the RNAseq data set (text is bold if p<0.05 after adjustment for multiple comparisons using the Benjamini method), and the running enrichment score calculated by GSEA software.

Fig 3: TUG controls PGC-1a protein abundance.a, WT and MTKO mice were treated by IP injection of insulin-glucose, or saline control, then sacrificed at the indicated times after injection. Quadriceps muscles were immunoblotted as indicated. b,c, WT and MTKO mice were treated by IP injection of insulin-glucose, or saline control, then sacrificed after 3 h. Lysates were prepared from quadriceps muscles, PGC-1a was immunoblotted, and the relative abundances in each sample were quantified using densitometry. Data in (c) are presented as mean ±SEM of biologically independent samples (N=3 in each group), analyzed using ANOVA with adjustment for multiple comparisons. d, WT and MTKO mice were treated with IP insulin-glucose, or saline control, then sacrificed 3 h later. RNA was prepared from quadriceps muscles, and Q-PCR was used to measure PGC-1a (Ppargc1a) mRNA abundance. Data are presented as mean ±SEM of biologically independent samples (N=3 in each group), analyzed using ANOVA with adjustment for multiple comparisons.

Fig 4: Effect of 8 weeks CR on AMPK activation, apoptosis, and mitochondrial biogenesis. (A): Representative Western blots and quantification of phosphorylation of AMPK (at Thr172), normalized to alpha AMPK, in the LV of sham or infarct rats, following 8 weeks of control or CR diets. (B): Representative Western blots and quantification of full-length and large fragment (17/19 kDa) of cleaved, activated caspase-3 in the LV of sham or infarct rats, following 8 weeks of control or CR diets. GAPDH served as loading control. (C): Representative Western blots and quantification of protein expression of Tfam, PGC-1alpha, mitochondrial components of complex IV (Cox 15 and I) and VDAC1, normalized to GAPDH, in LV tissue. (D): Relative copy number of mtDNA per diploid nuclear genome, as measured by real-time PCR. All data are mean ± SEM. n = 5–6 animals per group, *: p < 0.05; **: p < 0.01; ***: p < 0.001. Full size blots are provided in Supplementary Figure S2.

Fig 5: Effect of 8 weeks CR on AMPK activation, apoptosis, and mitochondrial biogenesis. (A): Representative Western blots and quantification of phosphorylation of AMPK (at Thr172), normalized to GAPDH, in WT and KO sham or infarct mice, following 8 weeks of control or CR diets. (B): Representative Western blots and quantification of full-length and cleaved, activated caspase-3. GAPDH served as loading control. (C): Representative Western blots and quantification of protein expression of Tfam, PGC-1alpha, mitochondrial components of complex IV (Cox 15 and I), and VDAC1, normalized to GAPDH, in LV tissue. All data are mean ± SEM. n = 6–8 animals per group, *: p < 0.05; **: p < 0.01; ***: p < 0.001. Full size blots are provided in Supplementary Figure S8.