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Atherosclerosis/cardiovascular disease are major causes of morbidity/mortality in obesity and type 2 diabetes (T2D), and have been associated with activation of innate immune cells, their diapedesis to the arterial intima and formation of the atherosclerotic plaque. While in obesity/T2D immune cell activation likely depends on dysregulated metabolism, the interaction between individual metabolic factors typical of these conditions (hyperglycemia, hyperlipidemia), innate immune cell activation, and the progression of atherosclerosis remains unclear. We, therefore, measured by flow cytometry cell surface expression of CD11b, CD14, CD16, CD62L, and CD66b, known markers of granulocyte (Gc) and monocyte (Mc) activation, in five healthy, five obese, and five T2D subjects, during 4-h i.v. infusions of 20% dextrose (raising blood sugar levels to ~220 mg/dL), 20% Intralipid (raising trygliceride levels to ~6 mmol/L), or a combination of the two. We hypothesized that both glucose and lipids would increase Gc/Mc surface marker expression, and simultaneous infusion would have an additive or synergistic effect. Surprisingly, though, infusion of glucose alone had little effect, while lipids, alone or combined with glucose, significantly increased expression of several markers (such as CD11b in Gc and Mc, and CD66 b in GC) within 60-90 min. Less pronounced increases in systemic inflammatory cytokines also occurred in obese and T2D subject, with no acute changes in gene expression of the the proinflammatory genes NFκB and CCR2. Our results suggest that lipids may be stronger acute contributors to innate cell activation than acute hyperglycemia per se, possibly helping shape more effective preventive dietary guidelines in T2D.

IntroductionCardiovascular complications are the leading cause of mortality in type 2 diabetes (T2DM), in which onset and progression of atherosclerosis is linked to chronic inflammation. Activation status of innate immune cells (granulocytes [Gc], monocytes [Mc]), as reflected by increased CD11b, CD66b, and other surface markers, increases their endothelial and cytokines/chemokines release. Whereas this inflammatory activation seems inversely related to poor glycemic control, the effect of acute spontaneous hyperglycemia on innate immune cell activation remains unclear.MethodsExpression of key markers (CD11b, CD14, CD16, CD62L, and CD66b) was therefore determined by flow cytometry on whole blood of healthy subjects and patients with T2DM with spontaneous fasting euglycemia or hyperglycemia both at baseline and after 30, 90, and 240 minutes of incubation at room temperature.ResultsHyperglycemic patients with T2DM had significantly higher Gc and Mc CD11b and Gc CD66b surface mean fluorescence intensity compared with the euglycemic patients with T2DM whose values were similar to those of the healthy controls. CD16 expression in CD14+CD16+ Mc was elevated in all patients with T2DM, regardless of glycemic levels.ConclusionOur data suggest that whereas the presence of diabetes per se may have a proinflammatory effect, hyperglycemia seems to further acutely exacerbate innate cell inflammatory status and their consequent endothelial adhesion and vascular damage potential.

Cardiovascular complications are the leading cause of mortality in type II diabetes (T2DM), in which onset and progression of atherosclerosis is linked to chronic inflammation. Activation status of innate immune cells (granulocytes Gc, monocytes Mc), as reflected by increased CD11b, CD66b and other surface markers, increases their endothelial and cytokines/chemokines release. While this inflammatory activation appears inversely related to poor glycemic control, the effect of acute spontaneous hyperglycemia on innate immune cell activation remains unclear. Expression of key markers (CD11b, CD14, CD16, CD62L, and CD66b) was therefore determined by flow cytometry on whole blood of healthy subjects and patients with T2DM with spontaneous, fasting eu- or hyper-glycemia both at baseline and after 30, 90, and 240 min. of room temperature incubation. Hyperglycemic patients with T2DM had significantly higher Gc and Mc CD11b and Gc CD66b surface mean fluorescence intensity (MFI) as compared to euglycemic patients with T2DM whose values were similar to healthy controls. CD16 expression in CD14+CD16+ Mc was elevated in all patients with T2DM, regardless of glycemic levels. Our data suggest that while the presence of diabetes per se may have a pro-inflammatory effect, hyperglycemia seems further acutely exacerbate innate cell inflammatory status, and their consequent endothelial adhesion and vascular damage potential.

Diet plays an important role in modulating exercise responses, including activation of the growth hormone (GH)/insulin-like growth factor-I (IGF-1) axis. Obesity and fat ingestion were separately shown to reduce exercise GH responses, but their combined effect, especially important in children, has not been studied. We therefore measured the GH response to exercise [30-min intermittent cycling, ten 2-min bouts at ∼80% maximal aerobic capacity (V̇o2max), separated by 1-min rest], started 45 min after ingestion of a high-fat meal (HFM) in 16 healthy [controls; body mass index percentile (BMI%ile) 51 ± 7], and 19 obese (Ob, BMI%ile 97 ± 0.4) children. Samples were drawn at baseline (premeal), and at start, peak, and 30 min postexercise. In the Ob group, a marked ∼75% suppression of the GH response (ng/ml) to exercise was observed (2.4 ± 0.6 vs. 10.6 ± 2.1, P < 0.001). This level of suppression was also significantly greater compared with age-, fitness-, and BMI-matched historical controls that had performed identical exercise in fasting conditions. Our data indicate that the reduction in the GH response to exercise, already present in obese children vs. healthy controls, is considerably amplified by ingestion of fat nutrients shortly before exercise, implying a potentially downstream negative impact on growth factor homeostasis and long-term modulation of physiological growth.

BackgroundAlthough altered metabolism has long been known to affect human breath, generating clinically usable metabolic tests from exhaled compounds has proven challenging. If developed, a breath-based lipid test would greatly simplify management of diabetes and serious pathological conditions (e.g., obesity, familial hyperlipidemia, and coronary artery disease), in which systemic lipid levels are a critical risk factor for onset and development of future cardiovascular events.MethodsWe, therefore, induced controlled fluctuations of plasma lipids (insulin-induced lipid suppression or intravenous infusion of Intralipid) during 4-h in vivo experiments on 23 healthy volunteers (12 males/11 females, 28.0 ± 0.3 years) to find correlations between exhaled volatile organic compounds and plasma lipids. In each subject, plasma triglycerides (TG) and free fatty acids (FFA) concentrations were both directly measured and calculated via individualized prediction equations based on the multiple linear regression analysis of a cluster of 4 gases. In the lipid infusion protocol, we also generated common prediction equations using a maximum of 10 gases.ResultsThis analysis yielded strong correlations between measured and predicted values during both lipid suppression (r = 0.97 for TG; r = 0.90 for FFA) and lipid infusion (r = 0.97 for TG; r = 0.94 for FFA) studies. In our most accurate common prediction model, measured and predicted TG and FFA values also displayed very strong statistical agreement (r = 0.86 and r = 0.81, respectively).ConclusionsOur results demonstrate the feasibility of measuring plasma lipids through breath analysis. Optimization of this technology may ultimately lead to the development of portable breath analyzers for plasma lipids, replacing blood-based bioassays.

BackgroundAlthough altered metabolism has long been known to affect human breath, generating clinically usable metabolic tests from exhaled compounds has proven challenging. If developed, a breath-based lipid test would greatly simplify management of diabetes and serious pathological conditions (e.g., obesity, familial hyperlipidemia, and coronary artery disease), in which systemic lipid levels are a critical risk factor for onset and development of future cardiovascular events.MethodsWe, therefore, induced controlled fluctuations of plasma lipids (insulin-induced lipid suppression or intravenous infusion of Intralipid) during 4-h in vivo experiments on 23 healthy volunteers (12 males/11 females, 28.0 ± 0.3 years) to find correlations between exhaled volatile organic compounds and plasma lipids. In each subject, plasma triglycerides (TG) and free fatty acids (FFA) concentrations were both directly measured and calculated via individualized prediction equations based on the multiple linear regression analysis of a cluster of 4 gases. In the lipid infusion protocol, we also generated common prediction equations using a maximum of 10 gases.ResultsThis analysis yielded strong correlations between measured and predicted values during both lipid suppression (r = 0.97 for TG; r = 0.90 for FFA) and lipid infusion (r = 0.97 for TG; r = 0.94 for FFA) studies. In our most accurate common prediction model, measured and predicted TG and FFA values also displayed very strong statistical agreement (r = 0.86 and r = 0.81, respectively).ConclusionsOur results demonstrate the feasibility of measuring plasma lipids through breath analysis. Optimization of this technology may ultimately lead to the development of portable breath analyzers for plasma lipids, replacing blood-based bioassays.

Obesity (Ob) and type 1 diabetes (T1DM) are associated with increased inflammation and oxidative stress, which are major pathogenetic pathways toward higher cardiovascular risks. Although long-term exercise protects against systemic inflammation and oxidation, acute exercise actually exerts pro-inflammatory and oxidative effects, prompting the necessity for better defining these molecular processes in at-risk patients; in particular, very little is known regarding obese and T1DM children. We therefore examined key inflammatory and oxidative stress variables during exercise in 138 peripubertal children (47 Ob, 12.7 ± 0.4 yr, 22 F, BMI% 97.6 ± 0.2; 49 T1DM, 13.9 ± 0.2 yr, 20 F, body mass index% [BMI] 63.0 ± 3.6; 42 healthy, CL, 13.5 ± 0.5 yr, 24 F, BMI% 57.0 ± 3.6), who performed 10 bouts of 2-min cycling ~80% VO(2max) , separated by 1-min rest intervals. Blood samples were drawn at baseline and peak exercise. Ob displayed elevated baseline interleukin-6 (IL-6, 2.1 ± 0.2 pg/mL, p < 0.005) vs. CL (1.5 ± 0.3), whereas T1DM displayed the greatest maximum exercise-induced change in IL-6 (1.2 ± 0.3) than in both Ob (0.7 ± 0.1, p < 0.001) and CL (0.6 ± 0.1, p < 0.0167). Myeloperoxidase (MPO) was elevated in T1DM (143 ± 30 ng/mL, p < 0.0167) vs. CL (89 ± 10) and Ob (76 ± 6), whereas increases in exercise only occurred in Ob and CL. Disparate baseline and exercise responses were also observed for 8-hydroxy-2'-deoxyguanosine, glutathione, and F(2) -isoprostane. This data show distinct patterns of dysregulation in baseline and adaptive immunologic and oxidative responses to exercise in Ob and T1DM. A full understanding of these alterations is required so that developing exercise regimens aimed at maximizing health benefits for specific dysmetabolic states can be achieved based on complete scientific characterization rather than empirical implementation.

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.

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.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.In Ob, TNF-a and IL-2 were significantly greater (p

Leukocytosis contributes to exercise-induced immune modulation, which is a mechanism of cardiovascular protection. However, this process is poorly defined in children. We therefore measured leukocytes in 45 healthy, 18 overweight, 16 type 1 diabetic, and 8 asthmatic children at pre, end-, and 30-min postexercise (30-min intermittent or 6-min continuous). In all groups, total leukocytes, neutrophils, lymphocytes, and monocytes increased at end-exercise, but returned to baseline by 30-min postexercise, including neutrophils, previously reported to remain elevated for at least some exercise formats. This highly preserved pattern indicates the importance of the adaptive response to physical stress across multiple health conditions. [ABSTRACT FROM AUTHOR]

Background Abnormal systemic concentrations of proinflammatory cytokines/chemokines have been implicated in the development of long-term cardiovascular complications in type 1 diabetes (T1DM) and obesity. Whether leukocyte white blood cell (WBC) gene expression of these proinflammatory mediators contributes to their increased systemic levels, however, remains unclear, especially in the pediatric patient populations. This study examines mRNA changes of 9 cytokines and chemokines in WBCs following ex vivo immunostimulation from 9 T1DM (13.4 ± 0.5 year, 4F/5 M), 23 overweight (OW, 12.3 ± 0.5 year, 10F/13M, BMI% 97.1 ± 0.5 and > 90.0), and 21 healthy (CL, 13.8 ± 0.7 year, 9F/12 M, BMI% 59.6 ± 4.6 and < 85.0) children. Methods All subjects had been maintained in euglycemic conditions for at least 90 min before blood draws. Whole blood was then sampled and incubated with anti-T-cell receptor (TCR) antibody or heat-aggregated IgG (HAG) to stimulate T-cell and Fc receptors (FcR), respectively. After lysis of leukocytes, mRNA levels of six tumor necrosis factor superfamily cytokines (TNFSF2, 5, 6, 7, 9, 14) and three chemokines (CCL8, 20, and CXCL10) were measured using RT-PCR. Results Following TCR stimulation, T1DM displayed significantly greater mRNA responses than CL for TNFSF5, 7, 9, and CCL8, and CXCL10; TNFSF9, CCL8, and CXCL10 were also significantly higher in T1DM than OW; no difference was observed between OW and CL. FcR stimulation induced similar responses across groups. Conclusions Leukocytes of T1DM children displayed exaggerated gene expression in response to ex vivo TCR induction of five key proinflammatory cytokines/chemokines. This elevated leukocyte gene expression may be one of the pathophysiological contributors to the development of vascular complications in T1DM. Copyright © 2009 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

The health effects of exercise depend on a complex equilibrium of often opposing mechanisms that may become altered in pathological states. In type 1 diabetes (T1DM), leukocyte activity dysregulation strongly correlates with diabetic vascular complications; it may therefore be important to identify alterations in exercise-induced leukocyte responses (total/fractional WBC counts, changes in leukocyte gene expression of immuno-modulatory factors). Little is known, however, about these responses, particularly in children. This study therefore investigated exercise-induced changes in leukocyte counts and mRNA expression of IL-6, IL- 10, VEGF, COX2, CD11a and HSP70 in control (n = 24, 14.1 ± 0.7 yrs, 8M/16F) and T1DM (n = 13, 13.3 ± 0.5 yrs, 8M/5F) children. Exercise consisted of 10 bouts of 2-min cycling at 80% VO2max separated by 1-min rest. For at least 90 min prior to and during exercise, euglycemia (80-110 mg/dL) was maintained for T1DM. Blood was drawn at baseline, end-exercise and 30-min post. mRNA expression was calculated as ratios of individual mRNAs and mRNA of the housekeeping protein β-actin to normalize for leukocytosis. The exercise-induced increase in WBC concentrations, observed in controls, was well preserved in T1DM. By 30-min post exercise, neutrophil and monocyte counts returned to baseline in both groups, while lymphocytes dipped below baseline. At end-exercise, WBC VEGF mRNA expression was significantly greater in T1DM than controls (p=0.041), while no difference was measured for other tested genes. Our data show that while the overall leukocytic response to exercise is well preserved in T1DM children, a significant alteration was present in the WBC mRNA expression of at least one major immune-modulator, VEGF, implicated in the pathological thickening of extracellular matrix causing reduced arterial perfusion. Our observations underscore the need for a deeper understanding of all aspects of exercise adaptation in populations at increased risk of long-term cardiovascular complications, including T1DM children. [ABSTRACT FROM AUTHOR]

Understanding the kinetic profiles of immunomodulatory factors during physical activity may help optimize the health benefits of exercise. This may be especially relevant for children with Type 1 Diabetes (T1DM), in which an imbalance of pro-/anti-inflammatory mediators that accelerates diabetic vascular complications may occur during/following exercise due to prior glycemic fluctuations. To date, exercise-induced alterations in immunomoduatory mediators in T1DM children remain largely unknown. This study examines 21 healthy (CNTRL, 13.9 ± 0.8 yrs, 8M/13F) and 21 diabetic (T1DM, 13.4 ± 0.3 yrs, 13M/8F) children. Blood glucose of T1DM was kept in the 80-110 mg/dL range for at least 90 min prior to and during exercise (ten 2-min cycling bouts at 80% VO2max, separated by 1-min intervals). Blood samples were drawn every second exercise bout, and at 15 and 30 min post exercise. In CNTRL, most tested mediators decreased during the first 15 min of exercise; some then increased slightly, remaining at or below baseline by end-exercise (the pro-inflammatory TNF-α, IL-12p70, GM-CSF, Eotaxin, MCP-1, and the anti-inflammatory IL-4); others clearly increased above baseline (IL-1α, IL-6, IL-8 IL-17, and MIP-1α, all pro-inflammatory/chemo-attractants). Conversely, in T1DM, the initial decrease was not observed, and most variables increased above baseline more markedly and more rapidly, resulting in significant differences in kinetic profiles vs CNTRL for TNF-α GM-CSF, Eotaxin, MCP-1, MIP-1α (10<0.05 - 0.001, AUC), and a significantly earlier time to peak values for IL-6, IL-17, and MIP-1α (p<0.05). The profile of the pro-inflammatory chemokine, IP-10, was similar between the 2 groups. During intense, intermittent exercise, the initial anti-inflammatory adaptation observed in age-matched healthy controls was absent in T1DM children. Further, the exercise-associated pro-inflammatory surge was greater in T1DM as compared to CNTRL. Our data suggest that optimization of exercise conditions may be crucial to maximize the health benefits of exercise in children with diabetes. [ABSTRACT FROM AUTHOR]

Altered leukocyte (WBC) activity, such as hypersecretion of pro-inflammatory cytokine/chemokines in type 1 diabetes (T1DM) has been implicated, in addition to early-stage accelerated damage to residual β cells, in the later onset and progression of vascular complications. Relatively little, however, is known about the regulation of WBC genes expressing pro-inflammatory molecules in response to immune stimuli in T1DM, particularly in children. Our study examines humoral and cell-mediated stimulation WBC cytokine/chemokine mRNA expression in T1DM and non-diabetic (nDM) children using a novel ex-vivo technique. Peripheral blood was drawn after at least 2 h of stable euglycemia in 14 T1DMs (13.2 ± 0.4 yrs, 8M/4F, +2 repeat subjects) and 26 nDMs (14.0 ± 0.6 yrs, 13M/13F). Blood was then incubated for 4 h with heat aggregated IgG (HAG) or anti-TCR antibody at body temperature to stimulate humoral and cell-mediated immune responses, respectively, with appropriate PBS and control IgG as comparisons, mRNA expression was then quantified, following WBC lysis, using a novel RT-PCR methodology developed by our group. Following TCR stimulation, most TNF super-family cytokines (TNFSFs) and CCL/CXCL chemokines displayed significantly increased WBC mRNA expression in T1DMs vs nDMs. Conversely, following HAG stimulation, no difference between groups was observed in for TNFSFs, various interleukins, or chemokines. Our data suggests that in T1DM children, WBC display an exaggerated proinflammatory response to immunologic stimuli that is limited to cell-mediated processes, while inflammatory responses to humoral stimulation appear intact. WBC cytokine expression may therefore contribute, at least in part, to the previously reported increased circulatory levels of pro-inflammatory cytokines in T1DM. While further investigations are needed to test the impact of this cell-mediated hyperactivity on the onset/progression of diabetic vascular complications, our findings may help identify new targets for specific, preventive anti-inflammatory strategies. [ABSTRACT FROM AUTHOR]