ASU Scholarship Showcase

We sought to evaluate the reproducibility of a liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based approach to measure the stable-isotope enrichment of in vivo-labeled muscle ATP synthase β subunit (β-F1-ATPase), a protein most directly involved in ATP production, and whose abundance is reduced under a variety of circumstances. Muscle was obtained from a rat infused with stable-isotope-labeled leucine. The muscle was homogenized, β-F1-ATPase immunoprecipitated, and the protein was resolved using 1D-SDS PAGE. Following trypsin digestion of the isolated protein, the resultant peptide mixtures were subjected to analysis by HPLC-ESI-MS/MS, which resulted in the detection of multiple β-F1-ATPase peptides. There were three β-F1-ATPase unique peptides with a leucine residue in the amino acid sequence, and which were detected with high intensity relative to other peptides and assigned with >95% probability to β-F1-ATPase. These peptides were specifically targeted for fragmentation to access their stable-isotope enrichment based on MS/MS peak areas calculated from extracted ion chromatographs for selected labeled and unlabeled fragment ions. Results showed best linearity (R[superscript 2] = 0.99) in the detection of MS/MS peak areas for both labeled and unlabeled fragment ions, over a wide range of amounts of injected protein, specifically for the β-F1-ATPase[subscript 134-143] peptide. Measured stable-isotope enrichment was highly reproducible for the β-F1-ATPase[subscript 134-143] peptide (CV = 2.9%). Further, using mixtures of synthetic labeled and unlabeled peptides we determined that there is an excellent linear relationship (R[superscript 2] = 0.99) between measured and predicted enrichment for percent enrichments ranging between 0.009% and 8.185% for the β-F1-ATPase[subscript 134-143] peptide. The described approach provides a reliable approach to measure the stable-isotope enrichment of in-vivo-labeled muscle β-F1-ATPase based on the determination of the enrichment of the β-F1-ATPase[subscript 134-143] peptide.

Background: Healthy individuals on the lower end of the insulin sensitivity spectrum also have a reduced gene expression response to exercise for specific genes. The goal of this study was to determine the relationship between insulin sensitivity and exercise-induced gene expression in an unbiased, global manner.

Methods and Findings: Euglycemic clamps were used to measure insulin sensitivity and muscle biopsies were done at rest and 30 minutes after a single acute exercise bout in 14 healthy participants. Changes in mRNA expression were assessed using microarrays, and miRNA analysis was performed in a subset of 6 of the participants using sequencing techniques. Following exercise, 215 mRNAs were changed at the probe level (Bonferroni-corrected P<0.00000115). Pathway and Gene Ontology analysis showed enrichment in MAP kinase signaling, transcriptional regulation and DNA binding. Changes in several transcription factor mRNAs were correlated with insulin sensitivity, including MYC, r=0.71; SNF1LK, r=0.69; and ATF3, r= 0.61 (5 corrected for false discovery rate). Enrichment in the 5’-UTRs of exercise-responsive genes suggested regulation by common transcription factors, especially EGR1. miRNA species of interest that changed after exercise included miR-378, which is located in an intron of the PPARGC1B gene.

Conclusions: These results indicate that transcription factor gene expression responses to exercise depend highly on insulin sensitivity in healthy people. The overall pattern suggests a coordinated cycle by which exercise and insulin sensitivity regulate gene expression in muscle.

Although insulin resistance in skeletal muscle is well-characterized, the role of circulating whole blood in the metabolic syndrome phenotype is not well understood. We set out to test the hypothesis that genes involved in inflammation, insulin signaling and mitochondrial function would be altered in expression in the whole blood of individuals with metabolic syndrome. We further wanted to examine whether similar relationships that we have found previously in skeletal muscle exist in peripheral whole blood cells. All subjects (n=184) were Latino descent from the Arizona Insulin Resistance registry. Subjects were classified based on the metabolic syndrome phenotype according to the National Cholesterol Education Program’s Adult Treatment Panel III. Of the 184 Latino subjects in the study, 74 were classified with the metabolic syndrome and 110 were without. Whole blood gene expression profiling was performed using the Agilent 4x44K Whole Human Genome Microarray. Whole blood microarray analysis identified 1,432 probes that were altered in expression ≥1.2 fold and P<0.05 after Benjamini-Hochberg in the metabolic syndrome subjects. KEGG pathway analysis revealed significant enrichment for pathways including ribosome, oxidative phosphorylation and MAPK signaling (all Benjamini-Hochberg P<0.05). Whole blood mRNA expression changes observed in the microarray data were confirmed by quantitative RT-PCR. Transcription factor binding motif enrichment analysis revealed E2F1, ELK1, NF-kappaB, STAT1 and STAT3 significantly enriched after Bonferroni correction (all P<0.05). The results of the present study demonstrate that whole blood is a useful tissue for studying the metabolic syndrome and its underlying insulin resistance although the relationship between blood and skeletal muscle differs.

Background: Although the effect of the fat mass and obesity-associated (FTO) gene on adiposity is well established, there is a lack of evidence whether physical activity (PA) modifies the effect of FTO variants on obesity in Latino populations. Therefore, the purpose of this study was to examine PA influences and interactive effects between FTO variants and PA on measures of adiposity in Latinos.

Results: After controlling for age and sex, participants who did not engage in regular PA exhibited higher BMI, fat mass, HC, and WC with statistical significance (P < 0.001). Although significant associations between the three FTO genotypes and adiposity measures were found, none of the FTO genotype by PA interaction assessments revealed nominally significant associations. However, several of such interactive influences exhibited considerable trend towards association.

Conclusions: These data suggest that adiposity measures are associated with PA and FTO variants in Latinos, but the impact of their interactive influences on these obesity measures appear to be minimal. Future studies with large sample sizes may help to determine whether individuals with specific FTO variants exhibit differential responses to PA interventions.

Introduction: Decreased insulin sensitivity blunts the normal increase in gene expression from skeletal muscle after exercise. In addition, chronic inflammation decreases insulin sensitivity. Chronic kidney disease (CKD) is an inflammatory state. How CKD and, subsequently, kidney transplantation affects skeletal muscle gene expression after exercise are unknown.

Methods: Study cohort: non-diabetic male/female 4/1, age 52±2 years, with end-stage CKD who underwent successful kidney transplantation. The following were measured both pre-transplant and post-transplant and compared to normals: Inflammatory markers, euglycemic insulin clamp studies determine insulin sensitivity, and skeletal muscle biopsies performed before and within 30 minutes after an acute exercise protocol. Microarray analyses were performed on the skeletal muscle using the 4x44K Whole Human Genome Microarrays. Since nuclear factor of activated T cells (NFAT) plays an important role in T cell activation and calcineurin inhibitors are mainstay immunosuppression, calcineurin/NFAT pathway gene expression was compared at rest and after exercise. Log transformation was performed to prevent skewing of data and regression analyses comparing measures pre- and post-transplant performed.

Result: Markers of inflammation significantly improved post-transplantation. Insulin infusion raised glucose disposal slightly lower post-transplant compared to pre-transplant, but not significantly, thus concluding differences in insulin sensitivity were similar. The overall pattern of gene expression in response to exercise was reduced both pre-and post-transplant compared to healthy volunteers. Although significant changes were observed among NFAT/Calcineurin gene at rest and after exercise in normal cohort, there were no significant differences comparing NFAT/calcineurin pathway gene expression pre- and post-transplant.

Conclusions: Despite an improvement in serum inflammatory markers, no significant differences in glucose disposal were observed post-transplant. The reduced skeletal muscle gene expression, including NFAT/calcineurin gene expression, in response to a single bout of exercise was not improved post-transplant. This study suggests that the improvements in inflammatory mediators post-transplant are unrelated to changes of NFAT/calcineurin gene expression.

Background: Physical activity (PA) interventions typically include components or doses that are static across participants. Adaptive interventions are dynamic; components or doses change in response to short-term variations in participant's performance. Emerging theory and technologies make adaptive goal setting and feedback interventions feasible.

Objective: To test an adaptive intervention for PA based on Operant and Behavior Economic principles and a percentile-based algorithm. The adaptive intervention was hypothesized to result in greater increases in steps per day than the static intervention.

Methods: Participants (N = 20) were randomized to one of two 6-month treatments: 1) static intervention (SI) or 2) adaptive intervention (AI). Inactive overweight adults (85% women, M = 36.9±9.2 years, 35% non-white) in both groups received a pedometer, email and text message communication, brief health information, and biweekly motivational prompts. The AI group received daily step goals that adjusted up and down based on the percentile-rank algorithm and micro-incentives for goal attainment. This algorithm adjusted goals based on a moving window; an approach that responded to each individual's performance and ensured goals were always challenging but within participants' abilities. The SI group received a static 10,000 steps/day goal with incentives linked to uploading the pedometer's data.

Results: A random-effects repeated-measures model accounted for 180 repeated measures and autocorrelation. After adjusting for covariates, the treatment phase showed greater steps/day relative to the baseline phase (p<.001) and a group by study phase interaction was observed (p = .017). The SI group increased by 1,598 steps/day on average between baseline and treatment while the AI group increased by 2,728 steps/day on average between baseline and treatment; a significant between-group difference of 1,130 steps/day (Cohen's d = .74).

Conclusions: The adaptive intervention outperformed the static intervention for increasing PA. The adaptive goal and feedback algorithm is a “behavior change technology” that could be incorporated into mHealth technologies and scaled to reach large populations.

Background: An evidence-based steps/day translation of U.S. federal guidelines for youth to engage in ≥60 minutes/day of moderate-to-vigorous physical activity (MVPA) would help health researchers, practitioners, and lay professionals charged with increasing youth’s physical activity (PA). The purpose of this study was to determine the number of free-living steps/day (both raw and adjusted to a pedometer scale) that correctly classified children (6–11 years) and adolescents (12–17 years) as meeting the 60-minute MVPA guideline using the 2005–2006 National Health and Nutrition Examination Survey (NHANES) accelerometer data, and to evaluate the 12,000 steps/day recommendation recently adopted by the President’s Challenge Physical Activity and Fitness Awards Program.

Methods: Analyses were conducted among children (n = 915) and adolescents (n = 1,302) in 2011 and 2012. Receiver Operating Characteristic (ROC) curve plots and classification statistics revealed candidate steps/day cut points that discriminated meeting/not meeting the MVPA threshold by age group, gender and different accelerometer activity cut points. The Evenson and two Freedson age-specific (3 and 4 METs) cut points were used to define minimum MVPA, and optimal steps/day were examined for raw steps and adjusted to a pedometer-scale to facilitate translation to lay populations.

Results: For boys and girls (6–11 years) with ≥ 60 minutes/day of MVPA, a range of 11,500–13,500 uncensored steps/day for children was the optimal range that balanced classification errors. For adolescent boys and girls (12–17) with ≥60 minutes/day of MVPA, 11,500–14,000 uncensored steps/day was optimal. Translation to a pedometer-scaling reduced these minimum values by 2,500 step/day to 9,000 steps/day. Area under the curve was ≥84% in all analyses.

Conclusions: No single study has definitively identified a precise and unyielding steps/day value for youth. Considering the other evidence to date, we propose a reasonable ‘rule of thumb’ value of ≥ 11,500 accelerometer-determined steps/day for both children and adolescents (and both genders), accepting that more is better. For practical applications, 9,000 steps/day appears to be a more pedometer-friendly value.

Background: Many studies used the older ActiGraph (7164) for physical activity measurement, but this model has been replaced with newer ones (e.g., GT3X+). The assumption that new generation models are more accurate has been questioned, especially for measuring lower intensity levels. The low-frequency extension (LFE) increases the low-intensity sensitivity of newer models, but its comparability with older models is unknown. This study compared step counts and physical activity collected with the 7164 and GT3X + using the Normal Filter and the LFE (GT3X+N and GT3X+LFE, respectively).

Findings: Twenty-five adults wore 2 accelerometer models simultaneously for 3Âdays and were instructed to engage in typical behaviors. Average daily step counts and minutes per day in nonwear, sedentary, light, moderate, and vigorous activity were calculated. Repeated measures ANOVAs with post-hoc pairwise comparisons were used to compare mean values. Means for the GT3X+N and 7164 were significantly different in 4 of the 6 categories (p < .05). The GT3X+N showed 2041 fewer steps per day and more sedentary, less light, and less moderate than the 7164 (+25.6, -31.2, -2.9 mins/day, respectively). The GT3X+LFE showed non-significant differences in 5 of 6 categories but recorded significantly more steps (+3597 steps/day; p < .001) than the 7164.

Conclusion: Studies using the newer ActiGraphs should employ the LFE for greater sensitivity to lower intensity activity and more comparable activity results with studies using the older models. Newer generation ActiGraphs do not produce comparable step counts to the older generation devices with the Normal filter or the LFE.

Background: Public parks can be an important setting for physical activity promotion, but to increase park use and the activity levels of park users, the crucial attributes related to active park use need to be defined. Not only user characteristics and structural park attributes, but also characteristics of the surrounding neighborhood are important to examine. Furthermore, internationally comparable studies are needed, to find out if similar intervention strategies might be effective worldwide. The main aim of this study was to examine whether the overall number of park visitors and their activity levels depend on study site, neighborhood walkability and neighborhood income.

Methods: Data were collected in 20 parks in Ghent, Belgium and San Diego, USA. Two trained observers systematically coded park characteristics using the Environmental Assessment of Public Recreation Spaces (EAPRS) tool, and park user characteristics using the System for Observing Play and recreation in Communities (SOPARC) tool. Multilevel multiple regression models were conducted in MLwiN 2.25.

Results: In San Diego parks, activity levels of park visitors and number of vigorously active visitors were higher than in Ghent, while the number of visitors walking and the overall number of park visitors were lower. Neighborhood walkability was positively associated with the overall number of visitors, the number of visitors walking, number of sedentary visitors and mean activity levels of visitors. Neighborhood income was positively associated with the overall number of visitors, but negatively with the number of visitors being vigorously active.

Conclusions: Neighborhood characteristics are important to explain park use. Neighborhood walkability-related attributes should be taken into account when promoting the use of existing parks or creating new parks. Because no strong differences were found between parks in high- and low-income neighborhoods, it seems that promoting park use might be a promising strategy to increase physical activity in low-income populations, known to be at higher risk for overweight and obesity.

Background: Plasma branched-chain amino acids (BCAA) are inversely related to insulin sensitivity of glucose metabolism in humans. However, currently, it is not known whether there is a cause-and-effect relationship between increased plasma BCAA concentrations and decreased insulin sensitivity.

Objective: To determine the effects of acute exposure to increased plasma BCAA concentrations on insulin-mediated plasma glucose turnover in humans.

Methods: Ten healthy subjects were randomly assigned to an experiment where insulin was infused at 40 mU/m2/min (40U) during the second half of a 6-hour intravenous infusion of a BCAA mixture (i.e., BCAA; N = 5) to stimulate plasma glucose turnover or under the same conditions without BCAA infusion (Control; N = 5). In a separate experiment, seven healthy subjects were randomly assigned to receive insulin infusion at 80 mU/m2/min (80U) in association with the above BCAA infusion (N = 4) or under the same conditions without BCAA infusion (N = 3). Plasma glucose turnover was measured prior to and during insulin infusion.

Results: Insulin infusion completely suppressed the endogenous glucose production (EGP) across all groups. The percent suppression of EGP was not different between Control and BCAA in either the 40U or 80U experiments (P > 0.05). Insulin infusion stimulated whole-body glucose disposal rate (GDR) across all groups. However, the increase (%) in GDR was not different [median (1st quartile – 3rd quartile)] between Control and BCAA in either the 40U ([199 (167–278) vs. 186 (94–308)] or 80 U ([491 (414–548) vs. 478 (409–857)] experiments (P > 0.05). Likewise, insulin stimulated the glucose metabolic clearance in all experiments (P < 0.05) with no differences between Control and BCAA in either of the experiments (P > 0.05).