Faculty Bibliography

Background: While fruit and vegetable (F&V) consumption is associated with reduced risk of stroke and coronary heart disease, there is little data on peripheral artery disease (PAD). We sought to study the association between F&V intake and prevalence of PAD in a large community cohort. Methods: From 2003 - 2008, over 3.5 million self-referred participants at >20,000 US sites completed a medical and lifestyle questionnaire and were evaluated by screening ankle-brachial index <0.9 for PAD. Subjects were queried for frequency of consumption of >3 servings of F&V (<1x/mo, 1x/mo- 1x/wk, 2-3x/wk, 4- 5x/wk, daily). Multivariate logistic regression analysis was used to estimate odds of PAD by F&V intake. Results: Among 3,523,545 individuals, mean age was 63.4 + 10.6 years and 63% were female. F&V intake ranged from 7% (<1x/month) to 29% (daily consumption of >3 servings). After adjustment for age, sex, race, smoking, physical activity, diabetes, hypertension, hyperlipidemia and family history of vascular disease, there was a step-wise inverse association of F&V intake with PAD (P for trend <0.0001; Figure). Compared to subjects with <1x/month consumption of >3 servings of F&V, daily intake was associated with 20% lower odds of PAD (OR 0.800, 95% CI 0.784 - 0.816). Conclusion: We present data from a large community cohort, in which F&V intake exhibited an inverse step-wise relationship with PAD prevalence after correction for multiple established risk factors, suggestive of protective effects in this vascular territory. (Figure Presented)

BACKGROUND: Chronic inflammation may contribute to insulin resistance (IR), metabolic syndrome and atherosclerosis although evidence of causality is lacking in humans. We hypothesized that very low-dose experimental endotoxemia would induce adipose tissue inflammation and systemic IR during a low-grade but asymptomatic inflammatory response and thus provide an experimental model for future tests of pharmacologic and genomic modulation of cardio-metabolic traits in humans. METHODS: Ten healthy, human volunteers (50% male, 90% Caucasian, mean age 22.7 +/- 3.8) were randomized in a double-masked, placebo-controlled, crossover study to separate 36-hour inpatient visits (placebo versus intravenous-LPS 0.6 ng/kg). We measured clinical symptoms via the McGill pain questionnaire and serial vital signs. Plasma and serum were collected for measurement of cytokines, C-reactive protein, insulin and glucose, serial whole blood & subcutaneous adipose tissue mRNA expression were measured by real-time PCR. HOMA-IR, a well-validated measure of IR was calculated to estimate insulin resistance, and frequently sampled intravenous glucose tolerance testing (FSIGTT) was performed to confirm an insulin resistant state. We performed ANOVA and within subject ANOVA to understand the differences in cytokines, adipose tissue inflammation and IR before and after LPS or placebo. RESULTS: There was no significant difference between placebo and LPS in clinical responses of symptom scores, body temperature or heart rate. However, low-dose endotoxemia induced a rapid and transient 25-fold induction of plasma TNF-alpha and 100-fold increase in plasma IL-6 (Figure 1B) (p < 0.001 for both) both peaking at two hours, followed by modest inflammation in adipose tissue with increases in mRNA levels of several inflammatory genes known to modulate adipose and systemic insulin resistance. Adipose tissue mRNA levels of IL-6 (peak 6-fold, ANOVA F = 27.5, p < 0.001) and TNF-alpha (peak 1.8-fold, F = 2.9, p = 0.01) increased with MCP-1 (peak 10-fold, F = 5.6, p < 0.01) and fractalkine (CX3CL1) (peak 15-fold, F = 13.3, p < 0.001). Finally, HOMA-IR was 32% higher following LPS compared to placebo (p < 0.01) and insulin sensitivity declined by 21% following LPS compared to placebo (p < 0.05). CONCLUSIONS: We present a low dose human endotoxemia model of inflammation which induces adipose tissue inflammation and systemic insulin resistance in the absence of overt clinical response. Such a model has the potential for broad and safe application in the study of novel therapeutics and genomic influences in cardio-metabolic disease.

Elevated plasma fibrinogen is a prothrombotic risk factor for cardiovascular disease (CVD). Recent small studies report that fibrinogen oxidative modifications, specifically tyrosine residue nitration, can occur in inflammatory states and may modify fibrinogen function. HDL cholesterol is inversely related to CVD and suggested to reduce the oxidation of LDL cholesterol, but whether these antioxidant functions extend to fibrinogen modifications is unknown. We used a recently validated ELISA to quantify nitrated fibrinogen during experimental human endotoxemia (N=23) and in a cohort of healthy adults (N=361) who were characterized for inflammatory and HDL parameters as well as subclinical atherosclerosis measures, carotid artery intima-medial thickness (IMT) and coronary artery calcification (CAC). Fibrinogen nitration increased following endotoxemia and directly correlated with accelerated ex vivo plasma clotting velocity. In the observational cohort, nitrated fibrinogen was associated with levels of CRP and serum amyloid A. Nitrated fibrinogen levels were not lower with increasing HDL cholesterol and did not associate with IMT and CAC. In humans, fibrinogen nitration was induced during inflammation and was correlated with markers of inflammation and clotting function but not HDL cholesterol or subclinical atherosclerosis in our modest sample. Inflammation-induced fibrinogen nitration may be a risk factor for promoting CVD events.

OBJECTIVE: Adipose inflammation plays a central role in obesity-related metabolic and cardiovascular complications. However, few human adipose-secreted proteins are known to mediate these processes. We hypothesized that microarray mRNA profiling of human adipose during evoked inflammation could identify novel adipocytokines. RESEARCH DESIGN AND METHODS: Healthy human volunteers (n = 14) were treated with intravenous endotoxin (3 ng/kg lipopolysaccharide [LPS]) and underwent subcutaneous adipose biopsies before and after LPS. On Affymetrix U133Plus 2.0 arrays, adipose mRNAs modulated >1.5-fold (with P < 0.00001) were selected. SignalP 3.0 and SecretomeP 2.0 identified genes predicted to encode secreted proteins. Of these, 86 candidates were chosen for validation in adipose from an independent human endotoxemia protocol (N = 7, with 0.6 ng/kg LPS) and for exploration of cellular origin in primary human adipocytes and macrophages in vitro. RESULTS: Microarray identified 776 adipose genes modulated by LPS; 298 were predicted to be secreted. Of detectable prioritized genes, 82 of 85 (96% [95% CI 90-99]) were upregulated (fold changes >1.0) during the lower-dose (LPS 0.6 ng/kg) validation study and 51 of 85 (59% [49-70]) were induced greater than 1.5-fold. Treatment of primary adipocytes with LPS and macrophage polarization to M1 proinflammatory phenotype increased expression by 1.5-fold for 58 and 73% of detectable genes, respectively. CONCLUSIONS: We demonstrate that evoked inflammation of human adipose in vivo modulated expression of multiple genes likely secreted by adipocytes and monocytes. These included established adipocytokines and chemokines implicated in recruitment and activation of lymphocytes, adhesion molecules, antioxidants, and several novel genes with unknown function. Such candidates may represent biomarkers and therapeutic targets for obesity-related complications.

The primary purpose of this study was to examine the effect of energy restriction on antioxidant capacity in trained athletes. Secondly, our study determined whether dietary protein source influenced the antioxidant response, performance, and immunity. Twenty male cyclists consumed either whey or casein supplement (40 g/day) in addition to their diet for 17 days. All subjects subsequently underwent 4 days of energy restriction using a formula diet (20 kcal/kg) while continuing protein supplementation. Energy restriction caused 2.7 +/- 0.3 kg weight loss, increased lymphocyte total glutathione (tGSH) 37%, red blood cell glutathione peroxidase 48%, plasma cysteine 12%, and decreased whole blood reduced to oxidized GSH (rGSH/GSSG) ratio by 52%. The only immunity factor altered by energy restriction was an increase in stimulated phagocytosis (65%). Acute submaximal exercise reduced blood tGSH but increased glutathione peroxidase. Performance of a high intensity cycle test following 45 min of moderate exercise tended to be reduced by energy restriction (P = 0.06) but was unaffected by protein source. Energy restriction caused a negative nitrogen balance with no difference from dietary protein source. In conclusion, acute energy restriction increased plasma cysteine and several markers of the glutathione antioxidant system in trained athletes. A high cysteine dietary protein source did not influence these responses.

The purpose of this study was to investigate the taste properties of protease inhibitors which are essential components of drug regimes used to treat human immunodeficiency virus (HIV) infection. In this study, the taste properties of four protease inhibitors (indinavir, ritonavir, saquinavir, and nelfinavir) were investigated in unmedicated HIV-infected patients and healthy controls. Three of the four protease inhibitors (indinavir, ritonavir, and saquinavir) were found to be predominantly bitter (with additional qualities of medicinal, metallic, astringent, sour, and burning). Nelfinavir was found to be relatively tasteless. HIV-infected and uninfected control subjects detected protease inhibitors at similar concentrations, but HIV-infected subjects perceived suprathreshold concentrations as more bitter than controls. Detection thresholds ranged from 0.0061 mM for saquinavir in HIV-infected patients to 0.0702 mM for ritonavir in uninfected control subjects. Suprathreshold studies indicated that protease inhibitors modified the taste perception of a variety of other taste compounds. These results are consistent with clinical findings that protease inhibitors produce taste complaints that can impact patient compliance.