Urocortin 3 activates AMPK and AKT pathways and enhances glucose disposal in rat skeletal muscle

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   Figure 1

   Expression of mUCN3 in rat skeletal muscle. Levels of (A) mouse Ucn3 mRNA expression in test and paired control tibialis cranialis muscles compared with a whole brain sample, measured using real-time PCR and (B) UCN3 peptide in test and control extensor digitorum longus muscles, measured by RIA, 1 week after IVE. Data are mean±s.e.m. (n=8). ***P<0.001 vs paired control.
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   Figure 2

   Expression of mUCN3 for 1 week resulted in increased muscle fibre size in the absence of a change in muscle mass, and no change in fibre type distribution. Muscle fibre diameter was measured in fixed transverse sections of TC muscles immunostained for laminin. Representative sections from (A) control and (B) mUCN3-expressing muscles are accompanied by (C) summary data. The percentage of each fibre type present in TC muscles was calculated after immunostaining of frozen transverse sections for type I, IIa and IIb fibres, with type IIx fibres indicated by lack of immunostaining. (D) Control and (E) mUCN3-expressing muscles immunostained for type I myosin heavy chain (MHC; red) and laminin (green). (F) Control and (G) mUCN3-expressing muscles immunstained for type IIa MHC (green) and type IIb MHC (red). (H) Summary fibre type distribution. Scale bars: 100 μm, n=6–8. Data are mean±s.e.m. *P<0.05 vs paired control. Black bars, control; white bars, UCN3 IVE muscle.
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   Figure 3

   Muscle UCN3 expression has contrasting effects on mediators of muscle hypertrophy and atrophy. Effects of local muscle UCN3 expression on relative mRNA and protein expression and phosphorylation of key mediators in pathways regulating muscle mass. Expression data were obtained by real-time PCR analysis of mRNA extracted from mUCN3-expressing and control TC muscles and are shown normalised to control. (A) mRNA levels of pro-hypertrophic genes: IGF1, IGF1 receptor (IGF1R), mighty (akirin1) and latent transforming growth factor-β 3 (LTBP3). (B) mRNA levels of pro-atrophic genes: myostatin, activin IIB receptor (activin 2BR), muscle ring finger protein 1 (MURF1), atrogin1 and the p65 subunit of nuclear factor κB (NFκB–p65). (C) pS256–FOXO1 and (D) p70S6k total protein levels and (E) sample immunoblots are shown. Total FOXO1 protein and pT389–p70S6k were not affected by forced UCN3 expression. C, control; T, test. Data are mean±s.e.m.; n=8; *P<0.05, **P<0.01, ***P<0.001 vs paired control.
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   Figure 4

   mUCN3 expression enhances muscle glucose disposal and increases protein levels of GLUT1 and GLUT4 glucose transporters. (A) Glucose uptake into paired TC muscles, estimated using 2-[1,2-³H(N)]-deoxy-d-glucose tracer during an intraperitoneal glucose tolerance test. Summary data for (B) GLUT1 and (C) GLUT4 protein contents quantified by western immunoblotting of mUcn3 expressing and paired control muscle lysates, normalised to control. (D) Sample immunoblots for GLUT1, GLUT4 and GAPDH, which was unaltered by the manipulation. Data are mean±s.e.m., n=8–9. *P<0.05, ***P<0.001 vs paired control.
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   Figure 5

   Enhanced muscle glucose disposal is associated with increased phosphorylation and protein levels of phosphoinositol 3-kinase pathway intermediates. Western immunoblotting for phosphorylation of regulatory residues and total protein content of PI3-kinase pathway intermediates was undertaken using mUCN3 expressing and paired control TC muscle lysates. Summary data for (A) pY612-IRS1, (B) total IRS1, (C) pS473–AKT, (D) total AKT, (E) pT642-AS160, (F) total AS160, (G) pS21–GSK3β and (H) total GSK3β are shown. (I) Sample immunoblots for each protein target and GAPDH, levels of which were unchanged by the manipulation, as were pS21–GSK3α and total GSK3α. Data are mean±s.e.m., n=8. *P<0.05, **P<0.01, ***P<0.001 vs paired control.
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   Figure 6

   Muscle mUCN3 expression increases activation of AMPK but decreases PGC1α. Western immunoblotting for (A) pT172–AMPK, (B) pS79–ACC, (C) total ACC and (D) total PGC1α protein was undertaken using mUCN3 expressing and paired control TC muscle lysates. Summary data and (E) sample immunoblots for each protein target and the loading control GAPDH are shown. Levels of total AMPK and GAPDH proteins were unchanged by the manipulation. Data are mean±s.e.m., n=8. *P<0.05, ***P<0.001 vs paired control.

• © 2014 The authors