Protein quality and distribution determines anabolic signaling, cellular energetics, and body composition

Building and maintaining muscle is critical to the quality of life for adults and elderly. Physical activity and nutrition are important factors for long-term muscle health. In particular, dietary protein – including protein distribution and quality – are under-appreciated determinants of muscle health for adults. The most unequivocal evidence for the benefit of optimal dietary protein at individual meals is derived from studies of weight management. During the catabolic condition of weight loss, higher protein diets attenuate loss of lean tissue and partition weight loss to body fat when compared with commonly recommended high carbohydrate, low protein diets. Muscle protein turnover is a continuous process in which proteins are degraded, and replaced by newly synthesized proteins. Muscle growth occurs when protein synthesis exceeds protein degradation. Regulation of protein synthesis is complex, with multiple signals influencing this process. The mammalian target of rapamycin (mTORC1) pathway has been identified as a particularly important regulator of protein synthesis, via stimulation of translation initiation. Key regulatory points of translation initiation effected by mTORC1 include assembly of the eukaryotic initiation factor 4F (eIF4F) complex and phosphorylation of the 70 kilodalton ribosomal protein S6 kinase (S6K1). Assembly of the eIF4F initiation complex involves phosphorylation of the inhibitory eIF4E binding protein-1 (4E-BP1), which releases the initiation factor eIF4E and allows it to bind with eIF4G. Binding of eIF4E with eIF4G promotes preparation of the mRNA for binding to the 43S pre-initiation complex. Consumption of the amino acid leucine (Leu) is a key factor determining the anabolic response of muscle protein synthesis (MPS) and mTORC1 signaling to a meal. Research from this dissertation demonstrates that the peak activation of MPS following a complete meal is proportional to the Leu content of a meal and its ability to elevate plasma Leu. Leu has also been implicated as an inhibitor of muscle protein degradation (MPD). In particular, there is evidence suggesting that in muscle wasting conditions Leu supplementation attenuates expression of the ubiquitin-proteosome pathway, which is the primary mode of intracellular protein degradation. However, this is untested in healthy, physiological feeding models. Therefore, an experiment was performed to see if feeding isonitrogenous protein sources with different Leu contents to healthy adult rats would differentially impact ubiquitin-proteosome (protein degradation) outcomes; and if these outcomes are related to the meal responses of plasma Leu. Results showed that higher Leu diets were able to attenuate total proteasome content but had no effect on ubiquitin proteins. This research shows that dietary Leu determines postprandial muscle anabolism. In a parallel line of research, the effects of dietary Leu on changes in muscle mass overtime were investigated. Animals consuming higher Leu diets had larger gastrocnemius muscle weights; furthermore, gastrocnemius muscle weights were correlated with postprandial changes in MPS (r=0.471, P<0.01) and plasma Leu (r=0.400, P=0.01). These results show that the effect of Leu on ubiquitin-proteosome pathways is minimal for healthy adult rats consuming adequate diets. Thus, long-term changes in muscle mass observed in adult rats are likely due to the differences in MPS, rather than MPD. Factors determining the duration of Leu-stimulated MPS were further investigated. Despite continued elevations in plasma Leu and associated translation initiation factors (e.g., S6K1 and 4E-BP1), MPS returned to basal levels ~3 hours after a meal. However, administration of additional nutrients in the form of carbohydrate, Leu, or both ~2 hours after a meal was able to extend the elevation of MPS, in a time and dose dependent manner. This effect led to a novel discovery that decreases in translation elongation activity was associated with increases in activity of AMP kinase, a key cellular energy sensor. This research shows that the Leu density of dietary protein determines anabolic signaling, thereby affecting cellular energetics and body composition.

Effect of proteolytic enzyme and fiber of papaya fruit on human digestive health

The World Health Organization (WHO, 2005) recommends consumption of fruits and vegetables as part of a healthy diet with daily recommendation of 5 servings or at least 400 g per day. Fruits and vegetables are good sources of vitamins, minerals, antioxidants, and fiber. Papaya fruit is known for his high nutrient and fiber content, and with few exceptions, it is generally consumed ripe due to its characteristic flavor and aroma. Digestion improvement has been attributed to consumption of papaya; this we speculate is attributed to the fiber content and proteolytic enzymes associated with this highly nutritious fruit. However, research is lacking that evaluates the impact of papaya fruit on human digestion. Papain is a proteolytic enzyme generally extracted from the latex of unripe papaya. Previous research has focused on evaluating papain activity from the latex of different parts of the plant; however there are no reports about papain activity in papaya pulp through fruit maturation. The activity of papain through different stages of ripeness of papaya and its capacity of dislodging meat bolus in an in vitro model was addressed. The objective of this study was to investigate whether papain activity and fiber content are responsible for the digestive properties attributed to papaya and to find a processing method that preserves papaya health properties with minimal impact on flavor. Our results indicated that papain was active at all maturation stages of the fruit. Ripe papaya pulp displayed the highest enzyme activity and also presented the largest meat bolus displacement. The in vitro digestion study indicated that ripe papaya displayed the highest protein digestibility; this is associated with proteolytic enzymes still active at the acidity of the stomach. Results from the in vitro fermentation study indicated that ripe papaya produced the highest amount of Short Chain Fatty Acids SCFA of the three papaya substrates (unripe, ripe, and processed). SCFA are the most important product of fermentation and are used as indicators of the amount of substrate fermented by microorganisms in the colon. The combination of proteolytic enzymes and fiber content found in papaya make of this fruit not only a potential digestive aid, but also a good source of SCFA and their associated potential health benefits. Irradiation processing had minimal impact on flavor compounds of papaya nectar. However, processed papaya experienced the lowest protein digestibility and SCFA production among the papaya substrates. Future research needs to explore new processing methods for papaya that minimize the detrimental impact on enzyme activity and SCFA production.

Characterization of the effect of nitrogen deficiency on above-ground plant phenotypes in relation to root complexity in maize

Nitrogen (N) is an essential plant nutrient in maize production, and if considering only natural sources, is often the limiting factor world-wide in terms of a plant’s grain yield. For this reason, many farmers around the world supplement available soil N with synthetic man-made forms. Years of over-application of N fertilizer have led to increased N in groundwater and streams due to leaching and run-off from agricultural sites. In the Midwest Corn Belt much of this excess N eventually makes its way to the Gulf of Mexico leading to eutrophication (increase of phytoplankton) and a hypoxic (reduced oxygen) dead zone. Growing concerns about these types of problems and desire for greater input use efficiency have led to demand for crops with improved N use efficiency (NUE) to allow reduced N fertilizer application rates and subsequently lower N pollution. It is well known that roots are responsible for N uptake by plants, but it is relatively unknown how root architecture affects this ability. This research was conducted to better understand the influence of root complexity (RC) in maize on a plant’s response to N stress as well as the influence of RC on other above-ground plant traits. Thirty-one above-ground plant traits were measured for 64 recombinant inbred lines (RILs) from the intermated B73 & Mo17 (IBM) population and their backcrosses (BCs) to either parent, B73 and Mo17, under normal (182 kg N ha-1) and N deficient (0 kg N ha-1) conditions. The RILs were selected based on results from an earlier experiment by Novais et al. (2011) which screened 232 RILs from the IBM to obtain their root complexity measurements. The 64 selected RILs were comprised of 31 of the lowest complexity RILs (RC1) and 33 of the highest complexity RILs (RC2) in terms of root architecture (characterized as fractal dimensions). The use of the parental BCs classifies the experiment as Design III, an experimental design developed by Comstock and Robinson (1952) which allows for estimation of dominance significance and level. Of the 31 traits measured, 12 were whole plant traits chosen due to their documented response to N stress. The other 19 traits were ear traits commonly measured for their influence on yield. Results showed that genotypes from RC1 and RC2 significantly differ for several above-ground phenotypes. We also observed a difference in the number and magnitude of N treatment responses between the two RC classes. Differences in phenotypic trait correlations and their change in response to N were also observed between the RC classes. RC did not seem to have a strong correlation with calculated NUE (ΔYield/ΔN). Quantitative genetic analysis utilizing the Design III experimental design revealed significant dominance effects acting on several traits as well as changes in significance and dominance level between N treatments. Several QTL were mapped for 26 of the 31 traits and significant N effects were observed across the majority of the genome for some N stress indicative traits (e.g. stay-green). This research and related projects are essential to a better understanding of plant N uptake and metabolism. Understanding these processes is a necessary step in the progress towards the goal of breeding for better NUE crops.