Journal: Molecular metabolism

Article Title: PGC-1β modulates catabolism and fiber atrophy in the fasting-response of specific skeletal muscle beds.

doi: 10.1016/j.molmet.2022.101643

Figure Lengend Snippet: Figure 1: PGC-1b expression is downregulated in skeletal muscle upon fasting. (A) Gene expression of PGC-1b and PGC-1a relative to TATA-box binding protein (TBP) in Quadriceps muscle of sedentary or exercised mice. (B and C) Gene expression of PGC-1b and PGC-1a relative to 18S in liver (B) and Gastrocnemius muscle (C) of ad-libitum fed or 24 h fasted mice. (D) Gene expression of PGC-1b relative to TATA-box binding protein (TBP) in different muscles and other tissues of control (WT) and PGC-1b muscle-specific knockout (MKO) mice. (E) Gene expression of PGC-1b, mitochondrial target genes and PGC-1a relative to TATA-box binding protein (TBP) in Gastrocnemius muscle of WT and MKO mice. (F and G) Protein levels of different mitochondrial chain complexes (F) in Gastrocnemius muscle of WT and MKO mice and representative immunoblots (G). As a loading control eukaryotic elongation factor 2 (eEF2) was used. (H and I) Quantification of (H) and representative succinate dehydrogenase (SDH) and cytochrome oxidase (COX) stainings of Gastrocnemius muscle cryo-sections (I) of WT and MKO mice. * indicates significant differences between sedentary and exercised mice, fed and fasted mice and WT and MKO mice; n ¼ 3e6.

Article Snippet: Forskolin stimulation Forty-eight hours post-transduction with HA-Flag-PGC-1b adenovirus, cells were serum starved for 1 h and next incubated with DMSO alone (control) or 10 mM Forskolin (Sigma, #F3917) in DMSO for 6 h. Subsequently, cells were collected for immunoblot analysis as described, using the following antibodies: PGC-1b (Proteintech, #22378-I-AP), eEF2 (Cell Signaling Technology, #2332S), p-CREBS133 (Cell Signaling Technology, #9198S) and CREB (Cell Signaling Technology, #9197S).

Techniques: Expressing, Gene Expression, Binding Assay, Muscles, Control, Knock-Out, Western Blot

Journal: Molecular metabolism

Article Title: PGC-1β modulates catabolism and fiber atrophy in the fasting-response of specific skeletal muscle beds.

doi: 10.1016/j.molmet.2022.101643

Figure Lengend Snippet: Figure 3: PGC-1b is necessary for the fasting-induced fiber atrophy. (AeC) Gastrocnemius (A), Soleus (B) and Tibialis anterior (TA) (C) absolute and relative muscle weights of ad-libitum fed or 24 h fasted mice. D-G) Minimal fiber ferrets (minFerret) of oxidative (D and E) and glycolytic (F and G) Gastrocnemius muscle cross-sections of ad-libitum fed (D and F) or 24 h fasted (E and G) mice. (H) Representative images of Gastrocnemius muscle cross-sections of ad-libitum fed or 24 h fasted WT and MKO mice. * indicates significant differences between WT and MKO mice; # indicates significant differences between fed and fasted conditions; n ¼ 4e6.

Article Snippet: Forskolin stimulation Forty-eight hours post-transduction with HA-Flag-PGC-1b adenovirus, cells were serum starved for 1 h and next incubated with DMSO alone (control) or 10 mM Forskolin (Sigma, #F3917) in DMSO for 6 h. Subsequently, cells were collected for immunoblot analysis as described, using the following antibodies: PGC-1b (Proteintech, #22378-I-AP), eEF2 (Cell Signaling Technology, #2332S), p-CREBS133 (Cell Signaling Technology, #9198S) and CREB (Cell Signaling Technology, #9197S).

Techniques:

Journal: Molecular metabolism

Article Title: PGC-1β modulates catabolism and fiber atrophy in the fasting-response of specific skeletal muscle beds.

doi: 10.1016/j.molmet.2022.101643

Figure Lengend Snippet: Figure 4: Transcriptomic patterns in MKO link modulated muscle proteostasis to fiber atrophy. (A) Gene expression changes in fed and fasted WT and MKO animals. A selection of Annotation Clusters indicates major predicted functional pathways that are altered only in the comparison of fed, in the overlap, and only in the comparison of fasted mice, respectively. (B) Gene expression comparison of the fasting response in WT and MKO mice. A selection of Annotation Clusters indicates major predicted functional pathways of physiological fasting that are dependent on muscle PGC-1b (WT only) and independent of muscle PGC-1b (overlap). The pathways found only in the comparison of MKO mice depict changes that arise de novo due to the absence of muscle PGC-1b in fasting. (C) Heatmaps of the relative expression of genes involved in protein synthesis and ribosome (left panel), and protein ubiquitination and proteasome (right panel). Colors represent the log2 fold change in gene expression compared to WT fed animals. (D) Gene expression of endogenous PGC-1b and PGC-1a relative to 18S in primary myotubes of WT mice treated with forskolin, 8-(4-chlorophenylthio)adenosine 30,50-cyclic monophosphate (CPT-cAMP) or 3-isobutyl-1-methylxanthine (IBMX) for 6 h (n ¼ 3). (E) Protein levels of overexpressed, epitope-tagged (HA) PGC-1b, endogenous p-CREBS133, endogenous CREB and endogenous eEF2 in primary myotubes of WT mice treated with forskolin for 6 h (n ¼ 3). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Forskolin stimulation Forty-eight hours post-transduction with HA-Flag-PGC-1b adenovirus, cells were serum starved for 1 h and next incubated with DMSO alone (control) or 10 mM Forskolin (Sigma, #F3917) in DMSO for 6 h. Subsequently, cells were collected for immunoblot analysis as described, using the following antibodies: PGC-1b (Proteintech, #22378-I-AP), eEF2 (Cell Signaling Technology, #2332S), p-CREBS133 (Cell Signaling Technology, #9198S) and CREB (Cell Signaling Technology, #9197S).

Techniques: Gene Expression, Selection, Functional Assay, Comparison, Expressing, Ubiquitin Proteomics

Journal: Molecular metabolism

Article Title: PGC-1β modulates catabolism and fiber atrophy in the fasting-response of specific skeletal muscle beds.

doi: 10.1016/j.molmet.2022.101643

Figure Lengend Snippet: Figure 5: Fasted MKO mice show reduced induction of myostatin, atrophy marker gene expression and protein ubiquitination. (A) Gene ontology (GO) analysis of differentially expressed (DE) genes between fasted MKO vs. fasted WT mice. (B) Gene expression of PGC-1b, myostatin (Mstn), muscle RING finger 1 (MuRF-1) and muscle atrophy F-box (MAFbx) relative to 18S in Gastrocnemius muscle of ad-libitum fed or 24 h fasted mice (n ¼ 5e6). (C and D) Representative immunoblot of ubiquitinylated proteins (C) in Gastrocnemius muscle of ad-libitum fed or 24 h fasted mice and corresponding quantification (D). Equal loading was verified by Ponceau staining of the membranes. (E) Gene expression of forkhead box O (Foxo) transcription factors 1, 3 and 4 relative to 18S in Gastrocnemius muscle of ad-libitum fed or 24 h fasted mice. (F and G) Representative immunoblots of total and phosphorylated forkhead box O 3a (Foxo3a) proteins in Gastrocnemius muscle of ad-libitum fed or 24 h fasted mice and corresponding quantifications (G). As a loading control eukaryotic elongation factor 2 (eEF2) was used. * indicates significant differences between WT and MKO mice; # indicates significant differences between fed and fasted conditions; n ¼ 3e6.

Article Snippet: Forskolin stimulation Forty-eight hours post-transduction with HA-Flag-PGC-1b adenovirus, cells were serum starved for 1 h and next incubated with DMSO alone (control) or 10 mM Forskolin (Sigma, #F3917) in DMSO for 6 h. Subsequently, cells were collected for immunoblot analysis as described, using the following antibodies: PGC-1b (Proteintech, #22378-I-AP), eEF2 (Cell Signaling Technology, #2332S), p-CREBS133 (Cell Signaling Technology, #9198S) and CREB (Cell Signaling Technology, #9197S).

Techniques: Marker, Gene Expression, Ubiquitin Proteomics, Western Blot, Staining, Control

Journal: Molecular metabolism

Article Title: PGC-1β modulates catabolism and fiber atrophy in the fasting-response of specific skeletal muscle beds.

doi: 10.1016/j.molmet.2022.101643

Figure Lengend Snippet: Figure 7: Nfatc1 activity is increased in fasted MKO animals. (A) ISMARA predictions of enriched transcription factor binding motifs in the comparison of fed and fasted WT and MKO mice, and the fasting response in WT and MKO animals. (B) Nuclear factor of activated T-cells, cytoplasmic 1 (Nfatc1) is the top ISMARA predicted motif from the gene expression profiles obtained in Gastrocnemius muscle of 24 h fasted WT and MKO mice. (C) Gene expression of Nfatc1 and predicted Nfatc1 target genes PGC-1a, protein-O-mannose kinase (Pomk), membrane associated ring-CH-type finger 1 (March1), SH3 domain containing kinase binding protein 1 (Sh3kbp1), methyltransferase like 11B (Mettl11b) and nitric oxide synthase 1 (Nos1) relative to 18S in Gastrocnemius muscle of ad-libitum fed or 24 h fasted mice. (D) Reporter gene assay using a 3 NFAT-luc plasmid, and co-transfection of NFATC1 alone or together with PGC-1b overexpression plasmids. (E and F) Representative immunoblots of total and phosphorylated Ca2þ/calmodulin-dependent protein kinase IIa (CaMKIIa) protein levels in Gastrocnemius muscle of ad-libitum fed or 24 h fasted mice (E) and corresponding quantifications (F). As a loading control eukaryotic elongation factor 2 (eEF2) was used. * indicates significant differences between WT and MKO mice; # indicates significant differences between fed and fasted conditions; n ¼ 3e6.

Article Snippet: Forskolin stimulation Forty-eight hours post-transduction with HA-Flag-PGC-1b adenovirus, cells were serum starved for 1 h and next incubated with DMSO alone (control) or 10 mM Forskolin (Sigma, #F3917) in DMSO for 6 h. Subsequently, cells were collected for immunoblot analysis as described, using the following antibodies: PGC-1b (Proteintech, #22378-I-AP), eEF2 (Cell Signaling Technology, #2332S), p-CREBS133 (Cell Signaling Technology, #9198S) and CREB (Cell Signaling Technology, #9197S).

Techniques: Activity Assay, Binding Assay, Comparison, Gene Expression, Membrane, Reporter Gene Assay, Plasmid Preparation, Cotransfection, Over Expression, Western Blot, Control