Title

The ubiquitin proteasome pathway in healthy, active adults: The effects of energy balance, endurance exercise, dietary leucine intake and intravenous leucine infusion on skeletal muscle protein breakdown

Date of Completion

January 2009

Keywords

Health Sciences, Nutrition|Health Sciences, Recreation

Degree

Ph.D.

Abstract

The ubiquitin proteasome pathway (UPP) represents the major intracellular pathway by which skeletal muscle proteins are degraded. The effects of dietary manipulations, exercise and leucine infusion on UPP component expression and activity, however, have not been characterized in humans. ^ Purpose. These studies describe the modulation of UPP component expression and activity in response to energy restriction, endurance exercise, alterations in dietary leucine intake and intravenous infusion of a leucine solution. ^ Methods. Study 1- Sixteen (11 men, 5 women) healthy, active adults participated in a two-phase, 20d diet intervention incorporating 10d eucaloric feeding followed by a 10d hypocaloric diet (∼80% energy needs). Participants were randomized to receive foods either High or Low in leucine content with protein intake set at 1.5 g·kg−1 ·d−1. Subjects underwent blood and muscle biopsy sampling following an acute bout of aerobic exercise (45min run @ 65% VO2peak) on day 7 of each diet phase, with a control Rest protocol completed on day 10 of each phase. ^ Study 2- Four healthy, active men participated in a 7d controlled feeding study, providing either 1.0 or 2.0g·kg −1·d−1 dietary protein, utilizing a crossover-design to evaluate the effects of an intravenous leucine infusion, versus saline. ^ In both studies, stable isotope infusions (Ring-2H 5-Phe and 15N-Phe) were used to measure fractional breakdown rate (FBR) of skeletal muscle via the tracee-release method. Chymotrypsin-like activity of the 26S proteasome's β5 subunits was assessed using a fluorescence-based assay. Expression of the proteasome subunit PSMA2, E3 ubiquitin ligase MAFbx and cysteine protease (Pro)Caspase-3 and activity of Caspase-3 were evaluated via standard Western blotting techniques. Plasma hormones and substrates were measured using HPLC, colorimetric assays and ELISAs. ^ Results. Study 1- In response to 10d energy restriction, subjects lost 1kg body weight (p<0.001) and experienced significant decreases in plasma glucose (p<0.001) and insulin (p=0.007) with increases in plasma lactate ( p=0.047) and preliminary FBR measures (p=0.005). When post-exercise data are evaluated in isolation, Caspase-3 (p=0.037) and 26S proteasome (p=0.032) activities increased significantly in response to negative energy balance. ^ Relative to rest, endurance exercise produced significant increases in plasma lactate (p<0.001), insulin (p=0.030) and cortisol (p<0.001) and expression of PSMA2 ( p=0.010) and Caspase-3 (p=0.038). Leucine intake was 1.6g·d−1 greater on the High versus Low diet (p=0.036) but did not alter any primary study variables. ^ Study 2- Leucine infusion, relative to saline, decreased PSMA2 (p=0.038) and MAFbx (p=0.082) expression and produced a nonsignificant decrease in 26S proteasome activity. ^ Conclusions. These data show increased expression of enzymes responsible for degrading muscle proteins in response to energy restriction, as well as increased activity of these enzymes during negative energy balance post-exercise. Accordingly, kinetic measures of muscle protein breakdown increased following energy restriction. Additionally, the use of intravenous leucine infusion as a means of mitigating UPP-mediated muscle catabolism shows promise. ^