Fig 2: Protein synthesis and mTOR signaling. (A) Mice tibialis anterior (TA) muscle were transduced with GFP control or Crtc2 expression vector. Crtc2 mRNA levels in the TA of mice fed ad libitum (Fed), or subject to three cycles of ADF (Fast) were isolated and weighted. N = 8 mice per group. (B) Quantitation of western blot (Supporting Information Figure S6B) for SUnSET protein synthesis assay. Mice electroporated and fed as in (A) were fasted overnight and treated with puromycin for 30 min and then the TA muscles collected for western blot. N = 4 mice per group. (C) TA muscle were isolated from mice electroporated and fed as in (A) for western blot analyses. N = 5 per group. (D–F) Quantitation of (C)

Fig 3: Crtc2 transgenic mice maintain enhanced glucose disposal and insulin tolerance. (A) Glucose tolerance test (GTT). Right panel, area under the curve. N = 10 mice per group. (B) Plasma insulin levels during GTT described in panel B. (C) Insulin tolerance tests. N = 10 mice per group. (D) Insulin-mediated uptake of 2-deoxy-D-glucose in primary myocytes transduced with adenovirus expressing Crtc2 or GFP control. N = 10 per group. Student’s t-test. (E) Crtc2 mice were treated +/− dox for 1 week and the gene induction compared by qPCR relative to the no dox groups. N = 10 mice per group. (F) Gene expression in primary myocytes transduced with adenovirus expressing Crtc2 or GFP control were compared by qPCR. N = 4 per group. (G) Myocytes stably expressing GFP, Crtc3 (lanes 2 and 3), dominant-negative Creb (A-CREB), or dominant-negative Crtc2 (dn-Crtc) were compared by western blot. See also Supporting Information Figure S3. (H) Quantitation of western blots shown in panel G. Arbitrary units (au). Data are mean ± SEM

Fig 4: Transcriptional control of fasting and weight loss and its regulation by Crtc2. (A) Mice tibialis anterior (TA) muscle were transduced with GFP control or Crtc2 expression vector. Crtc2 mRNA levels in the TA of mice fed ad libitum (Fed), or subject to three cycles of ADF (Fast) were compared by qPCR. N = 3 mice per group. (B) Venn diagram showing the numbers of DEGs identified by mRNA-seq comparing the effects of ADF in control versus Crtc2-transduced TA muscles. (C) Biological processes and functions regulated by ADF and Crtc2. Gene ontology (GO) analysis suggests that ADF regulates several processes/functions in a Crtc2-sensitive manner. The numbers of DEGs involved in each process are shown. See Supporting Information Dataset S1 for a complete list of represented GO annotations. (D) Examples of ADF-regulated transcriptional programs and mRNAs in control TA muscles. (E) Gene expression profiles in control and Crtc2-transduced TA muscles of mice subjected to ADF relative to the ad libitum fed mice (columns 1–2) , and effect of Crtc2 transduction relative to control during ADF (column 3). ADF-regulated genes in control muscle appear in bold. *Crtc2-regulated genes in mice subjected to ADF. *ADF-regulated genes in Crtc2-transduced muscle but not control. FC, fold change

Fig 5: Crtc2 expression in skeletal muscle enhances mitochondrial biogenesis. (A) Histological analysis of succinate dehydrogenase in myofibers from gastrocnemius muscle sections after dox treatment. Scale bar = 500 μm. (B) Oxidation of 3H-Palmitic acid in the supernatant of primary myocytes transduced with control, Crtc2, or Crtc3-expressing adenovirus. (C) Gene induction calculated relative to WT mice from isolated tibialis anterior muscles. N = 8 mice per group. (D) Gene expression in primary myocytes transduced with adenovirus expressing Crtc2 or GFP control 72-h post-transduction. (E) Crtc2 expression plasmid was electroporated into the tibialis anterior muscle and GFP plasmid in the contralateral leg. After 7 days, DNA was extracted for assessment of mitochondrial DNA (mt-DNA) for Crtc2/GFP control. N = 5 mice per group. *p < .05. Data are shown as mean ± SEM