Effect of adeno-associated virus (AAV)-mediated overexpression of PEPCK-M (Pck2) on Clenbuterol-induced muscle growth.

Abstract

We previously identified PEPCK-M (encoded by the Pck2 gene) to be highly up-regulated in skeletal muscle of pigs treated with Ractopamine, an anabolic beta-adrenergic receptor agonist. To determine whether PEPCK-M had a causative role in modulating the skeletal muscle growth response to Ractopamine, we used adeno-associated virus 1 (AAV1) to over-express Pck2 (AAV-Pck2) in murine skeletal muscle. A contralateral limb design was employed, such that each mouse served as its own control (injected with a GFP-only expressing AAV1, labelled AAV-GFP). Daily injections of Clenbuterol (1 mg/kg for 21 days) or vehicle control were also carried out to assess the effects of AAV-Pck2 overexpression on the anabolic response to a beta-adrenergic agonist. AAV-Pck2 overexpression in leg muscles of male C57BL6/J mice for 4 weeks (6-10 weeks of age) increased Pck2 mRNA (~100-fold), protein (not quantifiable) and enzyme activity (~3-fold). There was a trend (p = 0.0798) for AAV-Pck2 overexpression to reduce TA muscle weights, but there was no significant effect on muscle fibre diameters or myosin heavy chain isoform (MyHC) mRNA expression. When skeletal muscle growth was induced by daily administration of Clenbuterol (for 21 days), overexpression of AAV-Pck2 had no effect on the growth response, nor did it alter the expression of Phosphoserine Aminotransferase-1 (Psat1) or Asparagine Synthetase (Asns) mRNA or the Clenbuterol-induced decreases in MyHC IIa and IIx mRNA expression (p = 0.0065 and p = 0.0267 respectively). However AAV-Pck2 overexpression reduced TA muscle weights (p = 0.0434), particularly in the Control (vehicle treated) mice (p = 0.059 for AAV x Clenbuterol interaction) and increased the expression of Seryl-tRNA Synthetase (Sars) mRNA (p = 0.0477). Hence, contrary to the original hypothesis, AAV-Pck2 overexpression reduced TA muscle weights and did not mimic or alter the muscle hypertrophic effects of the beta-adrenergic agonist, Clenbuterol.