Title

Chocolate milk as a recovery beverage following endurance exercise: Impact on human skeletal muscle protein kinetics, intracellular signaling proteins, and proteins of the ubiquitin-proteasome pathway

Date of Completion

January 2010

Keywords

Health Sciences, Nutrition|Health Sciences, Recreation|Biology, Physiology

Degree

Ph.D.

Abstract

Amino acid provision via whole food consumption has been shown to enhance and suppress kinetic and molecular measures of human skeletal muscle protein (SMP) synthesis and breakdown, respectively. A variety of published data exist documenting the effects of feeding on synthesis and breakdown during post-endurance exercise recovery. However, no studies have comprehensively characterized both constituents of turnover during recovery from endurance exercise using a whole-food source of protein. ^ Purpose. The aim was to determine the effect of fat-free chocolate milk consumption on SMP fractional synthetic rate (FSR) and fractional breakdown rate (FBR) during recovery from an endurance exercise bout. Additionally, activity of intracellular signaling proteins (ISP) specific to protein translation and proteins in the ubiquitin-proteasome pathway was assessed. ^ Methods. Healthy, moderately trained, male runners (n=8) volunteered for the 2-wk study. Participants consumed a eucaloric diet that provided protein at 1.5 g·kg−1·d −1. On the 7th and 14th days of the study, participants completed exercise and blood/muscle sampling protocols. Each protocol consisted of an aerobic exercise bout (a 45-min run @ 65% VO 2peak immediately after which participants randomly consumed 480 mL of either fat-free chocolate milk (MILK) or an isocaloric, non-nitrogenous control beverage (CON). Blood was sampled and muscle biopsies taken from the participants following the exercise bout. ^ Stable amino acid isotope infusions (L-[Ring- 2H5]Phe and [15N]Phe) were used to measure FSR and FBR of the skeletal muscle protein via the direct incorporation method and tracee-release method, respectively. Chymotrypsin-like activity of the 26S proteasome β5 subunits was assessed using a fluorescence-based assay. Standard Western blotting techniques were used to determine phosphorylation of translationally-relevant proteins mTOR, Akt, eIF4E-BP1, rp-S6, eEF2, and FOXO3a; expression of the proteasome subunit PSMA2 and ubiquitin; and activity of the cysteine protease caspase-3. Plasma insulin was measured using an ELISA. ^ Results. FSR was greater during recovery in the MILK trial compared to CON (p=0.05). Spurious plasma [ 15N]Phe enrichment data prevented viable FBR data calculation. Phosphorylation of eIF4E-BP1 increased during recovery after consuming MILK (p=0.005), while there was no change after CON consumption. Akt phosphorylation decreased after consuming both MILK (p=0.003) and CON (p=0.005). FOXO3a phosphorylation decreased after CON consumption (p=0.004), but there was no change in the MILK trial. Activity of caspase-3 decreased following MILK consumption (p=0.004), while there was no activity change in the CON trial. Activity of the 26S proteasome core increased after CON consumption (p=0.004), while MILK maintained activity during recovery. There was no change in phosphorylation of mTOR, rp-S6, or eEF2 or total expression of PSMA2 or ubiquitin during recovery in either the MILK or CON trials. Plasma insulin at t=150 min (30 min into recovery) was greater in response to drinking MILK compared to CON (p=0.03). ^ Conclusions. These data show enhanced signaling of important translational proteins, suppression of proteolytic constituents, and increased synthesis of skeletal muscle protein during endurance exercise recovery in response to fat-free chocolate milk consumed at the onset of recovery. Athletes and individuals interested in optimizing post-exercise nutrient intake can consider fat-free chocolate milk as a practical and economical alternative when considering skeletal muscle repair. ^