TRB3 Regulates Protein Turnover in Mouse Skeletal Muscle
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Skeletal muscle atrophy is a critical condition associated with several diseases, including cancer cachexia, diabetes, and cardiac failure. Skeletal muscle mass loss is related to disrupted protein turnover involving increased protein degradation and suppressed protein synthesis. The IGF1/PI3K/Akt pathway has emerged as a major signaling cascade of protein turnover through mTOR (protein synthesis) and FOXOs (protein degradation) signaling, and is activated by anabolic signals such as insulin. Recently, TRB3, a mammalian homologue of Drosophila Tribbles 3, has been found in multiple tissues, including liver, fat, pancreas, and skeletal muscle, and negatively regulates Akt via direct binding to its phosphorylation sites (S473 and T308). Although increased TRB3 expression has been associated with insulin resistance in in vitro and in vivo models, its role in the regulation of skeletal muscle mass is not known. Here, we tested the hypothesis that TRB3 negatively regulates skeletal muscle mass in denervationinduced atrophy. We performed denervation of the sciatic nerve in one leg while the contralateral leg served as a control. At 9 days postdenervation, there was a robust increased in TRB3 mRNA (12.2fold) and protein expression (16.2fold) compared to the control. In addition, skeletal muscle weight was reduced by 2030% after 9 days of denervation, and insulinstimulated IRS1 Y612 (23%, P<0.01) and Akt T308 (22%, P<0.001) phosphorylation was attenuated. Musclespecific transgenic mice overexpressing TRB3 (TRB3TG) exhibited reduced skeletal muscle mass compared to wild type (TA; 10%, P=0.056, Gas; 20%, P=0.079), with substantially elevated mRNA expression of skeletal muscle specificubiquitin ligases, Atrogin1 (WT:1.000.33, TG: 1.60.16, A.U.; P=0.073) and MURF1 (WT: 1.000.1, TG: 1.70.2, A.U.; P<0.01). We then examined if skeletal muscle TRB3 overexpression would affect protein synthesis measured by assessing the incorporation of puromycin into nascent peptide chains. Muscle protein synthesis in TRB3TG mice was reduced by 60%. Grip strength was also decreased by 15% in TRB3TG mice when compared to wild type. However, musclespecific TRB3 overexpression did not accelerate or inhibit denervationinduced atrophy. In contrast, whole body TRB3 knockout mice (TRB3KO) displayed a 90% decrease in Atrogin1 protein expression compared to wild type (WT:1.000.03, KO: 0.10.01, A.U.; P<0.001). Furthermore, TRB3KO mice that received denervation for 9 days tended to prevent increasing of Atrogin1 (WT: 2.270.6; KO:1.390.07, A.U.; P=0.33) and MURF1 (WT: 2.60.5; KO: 1.720.23, A.U; P=0.093) protein expression compared to wild type. These data suggest that TRB3 could be a critical regulator of skeletal muscle mass under atrophic conditions and could be a target for therapeutic treatment.
