Stacy R. Oliver

Evidence for microRNA involvement in exercise-associated neutrophil gene expression changes

Exercise leads to a rapid change in the profile of gene expression in circulating neutrophils. MicroRNAs (miRNAs) have been discovered to play important roles in immune function and often act to attenuate or silence gene translation. We hypothesized that miRNA expression in circulating neutrophils would be affected by brief exercise. Eleven healthy men (19-30 yr old) performed 10, 2-min bouts of cycle ergometer exercise interspersed with 1-min rest at a constant work equivalent to approximately 76% of maximal oxygen uptake (Vo(2 max)). We used the Agilent Human miRNA V2 Microarray. A conservative statistical approach was used to determine that exercise significantly altered 38 miRNAs (20 had lower expression). Using RT-PCR, we verified the expression level changes from before to after exercise of seven miRNAs. In silico analysis showed that collectively 36 miRNAs potentially targeted 4,724 genes (2 of the miRNAs had no apparent gene targets). Moreover, when we compared the gene expression changes (n = 458) in neutrophils that have been altered by exercise, as previously reported, with the miRNAs altered by exercise, we identified three pathways, Ubiquitin-mediated proteolysis, Jak-STAT signaling pathway, and Hedgehog signaling pathway, in which an interaction of miRNA and gene expression was plausible. Each of these pathways is known to play a role in key mechanisms of inflammation. Brief exercise alters miRNA profile in circulating neutrophils in humans. These data support the hypothesis that exercise-associated changes in neutrophil miRNA expression play a role in neutrophil gene expression in response to physical activity.

Noninvasive measurement of plasma glucose from exhaled breath in healthy and type 1 diabetic subjects

Effective management of diabetes mellitus, affecting tens of millions of patients, requires frequent assessment of plasma glucose. Patient compliance for sufficient testing is often reduced by the unpleasantness of current methodologies, which require blood samples and often cause pain and skin callusing. We propose that the analysis of volatile organic compounds (VOCs) in exhaled breath can be used as a novel, alternative, noninvasive means to monitor glycemia in these patients. Seventeen healthy (9 females and 8 males, 28.0 ± 1.0 yr) and eight type 1 diabetic (T1DM) volunteers (5 females and 3 males, 25.8 ± 1.7 yr) were enrolled in a 240-min triphasic intravenous dextrose infusion protocol (baseline, hyperglycemia, euglycemia-hyperinsulinemia). In T1DM patients, insulin was also administered (using differing protocols on 2 repeated visits to separate the effects of insulinemia on breath composition). Exhaled breath and room air samples were collected at 12 time points, and concentrations of ~100 VOCs were determined by gas chromatography and matched with direct plasma glucose measurements. Standard least squares regression was used on several subsets of exhaled gases to generate multilinear models to predict plasma glucose for each subject. Plasma glucose estimates based on two groups of four gases each (cluster A: acetone, methyl nitrate, ethanol, and ethyl benzene; cluster B: 2-pentyl nitrate, propane, methanol, and acetone) displayed very strong correlations with glucose concentrations (0.883 and 0.869 for clusters A and B, respectively) across nearly 300 measurements. Our study demonstrates the feasibility to accurately predict glycemia through exhaled breath analysis over a broad range of clinically relevant concentrations in both healthy and T1DM subjects.

Sustained IL‐1α, IL‐4, and IL‐6 elevations following correction of hyperglycemia in children with type 1 diabetes mellitus

Objective:  An imbalance of pro‐/anti‐inflammatory cytokines may accelerate diabetic vascular complications and interfere with proper wound healing. Currently, limited available literature suggests that plasma concentrations of certain pro‐ and anti‐inflammatory cytokines may be altered during hyperglycemia/diabetes mellitus. It is still unclear, however, whether these concepts also apply to children with diabetes, and whether alterations in circulating cytokine levels are a permanent feature of diabetes or an acute effect of fluctuating glucose concentrations.

Effects of Exercise on microRNA Expression in Young Males Peripheral Blood Mononuclear Cells

MicroRNAs are increasingly seen as targets of drug discovery because they influence gene function acting both to silence and subtly modulate protein translation. Little is known about effects of dynamic physiological states on microRNA regulation in humans. We hypothesized that microRNA expression in peripheral blood mononuclear cells (PBMCs) would be affected by brief exercise. Twelve young men performed brief bouts of heavy exercise. PBMC microRNA was analyzed before and immediately after exercise using the Agilent Human microRNA V2 Microarray. Exercise altered expression level of 34 microRNAs (FDR < 0.05). Many of them play roles in inflammatory processes (e.g., miR‐125b[↓], down‐regulated by proinflammatory factor LPS; and miR‐132[↑], 125b[↓] and let‐7e[↓] involved inTLR4 signaling). Using previous exercise data in PBMCs, we linked the microRNA changes to specific gene pathways. This analysis identified 12 pathways including the TGF‐β and MAPK signaling. We also compared exercise‐associated microRNA changes in PBMCs with the exercise‐associated microRNAs previously identified in neutrophils. Nine microRNAs were affected in both PBMCs and neutrophils, but only six changed in the same direction. A commonly occurring physiologic perturbation, brief heavy exercise, changes microRNA profiles in PBMCs, many of which are related to inflammatory processes. The pattern of change suggests that exercise differentially influences microRNAs in leukocyte subtypes. Clin Trans Sci 2012; Volume #: 1–7

Increased oxidative stress and altered substrate metabolism in obese children

OBJECTIVE Pediatric obesity, a major risk factor for cardiovascular diseases and diabetes, has steadily increased in the last decades. Although excessive inflammation and oxidation are possible biochemical links between obesity and cardiovascular events in adults, little information is available in children. Furthermore, effects of gender and fitness on the interaction between dyslipidemia and oxidative/inflammatory stress in children are mostly unknown. METHODS Therefore, we measured systemic markers of oxidation (F(2)-isoprostanes [F(2)-IsoP] and antioxidants) and inflammation (interleukin-6 [IL-6] and leukocyte counts) and metabolic variables in 113 peripubertal children (55 obese [Ob] age and gender-adjusted BMI% ≥ 95(th), 25 Females [F]; 15 overweight [OW] BMI% 85(th)-95(th), 8 F; 43 normoweight [NW] 25 F). RESULTS When compared with NW, Ob displayed elevated F(2)-IsoP (99 ± 7 vs. 75 ± 4 pg/mL, p<0.005), IL-6 (2.2 ± 0.2 vs. 1.5 ± 0.3 pg/mL, p<0.005), elevated total leukocytes and neutrophils, altered levels of total cholesterol , low- and high-density-lipoprotein cholesterol, triglycerides, free fatty acids, glucose, and insulin (all p<0.005). This pattern was present in both genders and over a broad range of fitness in Ob. CONCLUSIONS Our data indicate that alterations in metabolic control and a concomitant increase in inflammation and oxidative stress occur early in life in obese children, likely exposing both genders to a similar degree of increased risk of future cardiovascular diseases.

Aspects of Inflammation and Oxidative Stress in Pediatric Obesity and Type 1 Diabetes: An Overview of Ten Years of Studies

Obesity and type 1 diabetes (T1DM) are the two most common conditions of altered metabolism in children and adolescents. In both, similar long-term cardiovascular complications are known to occur, mediated in large part by underlying inflammatory and oxidative processes whose biochemical details remain relatively unclear. Through a series of experiments in these patient populations, over the last decade our laboratory has clarified a number of key issues in this field. Interestingly, while obese and type 1 diabetic children often differed in the specific type and magnitude of molecular alterations, in both groups a clear exaggeration of inflammatory and oxidative activation was detected when compared to healthy, age-matched controls. Our main findings include definition of resting and exercise-induced cytokine patterns and leukocyte profiles, patterns of activation of immune cells in vitro, and correlation of the magnitude of observed alterations with severity of obesity and quality of glycemic control. Further, we have identified a series of alterations in growth factor profiles during exercise that parallel inflammatory changes in obese children. This paper offers a concise overview of the salient results from this decade-long research effort.

Altered inflammatory, oxidative, and metabolic responses to exercise in pediatric obesity and type 1 diabetes

Rosa JS, Oliver SR, Flores RL, Ngo J, Milne GL, Zaldivar FP, Galassetti PR. Altered inflammatory, oxidative, and metabolic responses to exercise in pediatric obesity and type 1 diabetes.

Inflammatory Cytokine Profiles During Exercise in Obese, Diabetic, and Healthy Children

Objective: Modulation of inflammatory status is considered a key component of the overall health effects of exercise. This may be especially relevant in children with obesity (Ob) or type 1 diabetes (T1DM), in which an imbalance between pro- and anti-inflammatory mediators could accelerate onset and progression of cardiovascular complications. To date, exercise-induced alterations in immuno-modulatory mediators in Ob and T1DM children remain largely unknown. Methods: In this study, we monitored the kinetic profiles of 8 pro-and anti-inflammatory cytokines (TNF-a, IL-6, IL-2, IL-8, IL-5, IL-13, IL-10, IL-4) during a standardized exercise challenge (ten 2-min cycling bouts at 80% VO2max, separated by 1-min intervals) in 23 Ob (12 females, 11 males), 23 T1DM (10 females and 13 males) patients and 20 healthy (CL, 10 females and 10 males) children. Blood glucose of T1DM patients was kept in the 4.4-6.1 mM range for at least 90 minute prior to and during exercise. Blood samples were drawn at rest and after every other exercise bout. Results: In Ob, TNF-a and IL-2 were significantly greater (p<0.0167) as compared to T1DM and CL, both at baseline and throughout exercise. All other variables, while not significant, were quantitatively elevated in Ob vs. CL. In T1DM, IL-4 and IL-8 levels were similar to Ob, IL-2 and TNF-a similar to CL, and IL-6, IL-5, IL-13, IL-4 levels were intermediate between the Ob and CL groups. Conclusions: During exercise, therefore, both Ob and T1DM children displayed exaggerated pro-inflammatory responses, although with clearly different magnitude and involved mediators. Our data support the necessity to identify specific exercise formats through which each at-risk pediatric population can draw maximal beneficial health effects. Conflict of interest:None declared.

Resting and exercise-induced IL-6 levels in children with Type 1 diabetes reflect hyperglycemic profiles during the previous 3 days

Poor glycemic control in Type 1 diabetes (T1DM) causes long-term cardiovascular complications, at least in part via chronic, low-grade inflammation associated with recurrent hyperglycemia. While physical activity can reduce both inflammation and cardiovascular risks, the underlying molecular mechanisms remain unclear. This is particularly important for T1DM children, for whom the prevention of long-term cardiovascular complications must include optimization of exercise-related anti-inflammatory strategies. We therefore studied the effect of prior hyperglycemia on resting and exercise-induced inflammatory status (plasma IL-6) in T1DM children. Glycemia was continuously recorded with a continuous glucose monitoring system (CGMS) system for 63 h preceding a 30-min intermittent cycling exercise protocol at approximately 80% peak rate of oxygen uptake (VO2max). Euglycemia (4.4-6.1 mM) was maintained for 90 min before, during, and 30 min after exercise. IL-6 plasma concentration (pg/ml) was measured at baseline, at end exercise, and 30 min postexercise. Subjects were then divided into quartiles based on average glycemia during the CGMS recording. IL-6 levels (pg/ml) were lowest in the quartile with lowest average 3-day glycemia and increased proportionally to greater hyperglycemic exposure; this was observed at baseline (0.86 +/- 0.10, 1.06 +/- 0.16, 1.14 +/- 0.14, 1.20 +/- 0.16), absolute IL-6 change (Delta) at end exercise (0.20 +/- 0.16, 0.32 +/- 0.10, 0.48 +/- 0.09, 0.62 +/- 0.13), and Delta at 30 min postexercise (0.49 +/- 0.13, 0.71 +/- 0.16, 0.89 +/- 0.14, 1.38 +/- 0.33). Therefore, poorly controlled glycemic profile, even in the 63 h preceding an exercise challenge, can alter inflammatory adaptation in T1DM children. Our data underscore the necessity to fully understand all molecular aspects of physical activity to provide the scientific rationale for exercise regimens that will be able to maximize health benefits for T1DM children.

Altered Kinetics of Interleukin-6 and Other Inflammatory Mediators During Exercise in Children With Type 1 Diabetes

Background Leukocyte mobilization and secretions of cytokines, chemokines, and growth factors in children during exercise are necessary biochemical signals for physiological growth and long-term cardiovascular protection. Because of glycemic instability, altered exercise responses, particularly the proinflammatory cytokine interleukin (IL)-6, may occur in type 1 diabetes mellitus (T1DM) that could influence the onset/progression of diabetic vascular complications. Relatively little is known, however, on most molecular aspects of immunomodulatory adaptation to exercise in diabetic children. Methods We therefore studied 21 children (age, 13.4 ± 0.3 years; 13 boys/8 girls) with T1DM and 21 age-matched healthy controls during 30 minutes of intense and intermittent cycling exercise. Euglycemia was maintained during and for greater than 90 minutes before exercise; blood samples for IL-6 and other cytokines/chemokines were drawn before, during (every 6 minutes), and after (every 15 minutes) exercise. Results In T1DM, exercise-induced IL-6 peak occurred earlier and with greater magnitude than that in controls; an exploratory analysis of additional inflammatory mediators displayed a similarly accelerated/exaggerated pattern in T1DM, including the kinetic profiles of tumor necrosis factor α, IL-4, IL-12p70, IL-17, granulocyte-monocyte colony-stimulating factor, monocyte chemoattractant protein-1, macrophage inflammatory protein-1α, and eotaxin (interferon-inducible protein-10 was the only measured variable essentially indistinguishable between groups). Conclusion Therefore, during intense and intermittent exercise, significant alterations in the immunologic pattern of inflammatory regulation occurred in children with T1DM as compared with healthy controls. Our findings underscore how the understanding of all the underlying molecular mechanisms is a necessary prerequisite for achieving effective use of exercise and the full manifestation of its health benefits, particularly in understudied populations such as children with T1DM who are at increased risk for cardiovascular complications.