Monocytes possess constitutively active caspase-1 and thus can release IL-1b if the expression of pro-IL-1b is induced by the activation of TLRs . Thus, saturated FAs in plasma FFAs may be sufficient to induce inflammasome mediated IL-1b release, a hallmark of monocyte activation. Activated monocytes transmigrate into peripheral tissues and are differentiated into macrophages that generate inflammatory signals. Therefore, alleviation of monocyte activation by dietary constituents may be an effective strategy to suppress the triggering of inflammation that leads to enhanced inflammation in peripheral tissues. In this study, we determined whether changes in plasma FFA concentrations induced by a single moderately high-fat breakfast modulate plasma cytokine concentrations and the propensity of monocyte activation in blood and whether supplementation with blueberry powder that contains anti-inflammatory polyphenols inhibits these processes.This investigation conforms to the principles outlined in the Declaration of Helsinki and was approved by the Institutional Review Board for Human Subjects at the University of California Davis. Written informed consent was obtained from all subjects who were recruited from the greater Sacramento, California area. The health screening and study visits were conducted at the Western Human Nutrition Research Center in Davis, California. This trial was registered at clinicaltrials.gov as NCT01594008. Inclusion criteria included: 1) age between 18 and 60 y and 2) a normal BMI . Exclusion criteria included: 1) total blood cholesterol >240 mg/dL, 2) TGs >300 mg/dL, 3) hemoglobin <11.5 mg/dL, 4) abnormal results in clinical chemistry and hematology panels, 5) inflammatory or metabolic diseases, 6) use of non-steroidal anti-inflammatory drugs including asthma and allergy medications, 7) unwillingness to discontinue use of dietary supplements before and during the study period, and 8) vegetarianism.
In total, 27 subjects participated in the study ; 4 subjects did not complete the study .A schematic of the study design is depicted in Figure 2. Subjects were blinded to the dose of BB powder they were given. Researchers handling samples and data did not know what dose of BB powder subjects received on test day 2 or 3. The frozen BB yogurt supplemented with either placebo control or BB powder was coded by letter,greenhouse snap on clamp and samples were coded by test day. The provision of the placebo control powder on the first day to all subjects was done to mitigate potential carryover effects of the BB powder. The registered dietitian coded the different treatments . The randomization scheme was generated by using the website Randomization.com s ‘‘second generator.’’ The registered dietitian created a randomized list of the 2 and 4 servings codes and assigned subjects sequentially as subject numbers were assigned. If a subject dropped out after being randomized, the next subject was assigned to the treatment schedule of the dropped subject. Subjects were instructed by a registered dietitian to follow a low polyphenol and low-omega-3 FA diet and limit consumption of fruits, vegetables, soy, fatty fish , whole grains, nuts, coffee, tea, and chocolate starting 3 d before each test day. The night before each test day, subjects consumed a standardized dinner provided by the WHNRC that included a burrito as well as yogurt and lemonade to minimize masking effects of previous diet and variations in fasting levels of endpoints caused by the different dinners consumed by the subjects . On each test day, subjects arrived at the WHNRC after a 12-h overnight fast. Subjects had their body temperature, blood pressure, and weight measured and then had blood drawn. Subjects consumed the breakfast meal with either placebo control or BB powder prepared in yogurt as described below under the test meals section. Subjects were given 20 min to consume the entire breakfast, after which they were instructed not to eat or drink anything other than water and were allowed to return to their normal daily activities.
Subjects returned to the WHNRC 3.5 h after consumption of the test meal for a postprandial blood draw. Postprandial peaks of the plasma concentration of TGs occur on average 3.5 h after the consumption of a high-fat breakfast . Following the postprandial blood draw, subjects were allowed to return to their normal dietary habits until 3 d before their next test day. Subjects consumed the placebo control powder on their first test day. On the test days 2 and 3, subjects consumed varying amounts of the BB powder that were equivalent to 2 or 4 servings of fresh BBs in a random order.The MHF breakfast contained 650 kcal and met the following nutrient specifications: 40% kcal derived from fat, primarily from food sources containing long chain FAs ; 15% kcal derived from protein; and 45% kcal derived from carbohydrates . The MHF breakfast consisted of a bagel sandwich and sucrose-sweetened yogurt, which served as the vehicle for administering the BB or placebo control powder. Table 1 presents the nutrition information separately for the base MHF meal with and without the addition of the placebo control/BB powder,which provided an additional 191 kcal. The addition of the control/BB powder decreased the percentage of fat content of the overall meal from 40% to 31.5%. The amount of total fat in grams remained the same including a small amount of fat from the BBs and the matched control powder for each test meal. The placebo control and BB powder mixed in yogurt were prepared in advance and frozen, and each of them was served in a separate cup, so that each subject consumed all of the BB or placebo control powder. The rationale for doing so was to realistically simulate the consumption of BBs with an MHF meal while maintaining the same nutrient composition of each test meal. A registered dietitian designed the pretest dinner and the MHF breakfast by using the Nutrient Data System for Research software version 2011, developed by the Nutrition Coordinating Center of the University of Minnesota in Minneapolis, Minnesota and ProNutra software. The BB powder was provided by the US high bush Blueberry Council and was composed of a 50/50 mixture of 2 varieties of high bush BBs, Tifblue and Rubel . Whole BBs were freeze-dried, milled, and stored in sealed aluminum cans with a desiccant at 220 C. The placebo control powder and 2 different servings [2 and 4 servings] of BB powder preparations were made to match: color , flavor , fat , carbohydrates , protein , and fiber , and vitamin C of the preparation containing 4 servings of BB powder .
Nutrient content was confirmed by chemical analysis at Medallion Labs.Humans spend the majority of the day in the postprandial state. Increased plasma TGs, insulin surge, and accompanying decrease in plasma FFAs are general features of immediate postprandial changes in blood . Decreased postprandial plasma FFA concentrations are generally preceded by a meal induced postprandial insulin surge, which suppresses lipolysis and stimulates re-esterification of FFAs into TGs. Whether and how plasma concentrations of FFAs modulate postprandial inflammation are not clear. Saturated FAs can activate TLR2 and TLR4 leading to the expression of markers of inflammation in cell culture systems . TLR2- or TLR4-deficient mice were protected from high saturated fat diet-induced inflammation and insulin resistance , suggesting that saturated FAs derived from dietary fat can induce TLR-mediated inflammation. The concentrations of plasma FFAs and TGs are temporally regulated in concert with endocrine changes in fasting and postprandial states. Our previous mechanistic study showed that both exogenous palmitic acid and endogenous FFAs, hydrolyzed by added LPL from TGRLs isolated from human subjects who consumed a high fat meal, induce the activation of TLR2 in primary blood monocytes and whole blood . TLR2 activation was assessed by the receptor dimerization and the expression of the monocyte specific TLR2/inflammasome-mediated IL-1b as a biochemical readout. Treatment of primary monocytes or whole blood with LPL also caused increased expression of IL-1b, providing a mechanistic insight for the causal role of plasma FFAs on cytokine production . Together, these results suggest that the concentration of plasma FFAs is an important determinant for the expression of TLR target gene products in blood monocytes and possibly other blood leukocytes that express TLR2 or TLR4. The results of our current study show that the concentrations of cytokines decreased in 3.5-h postprandial plasma after the MHF breakfast as the concentrations of FFAs declined compared with those of fasting plasma , suggesting that eating breakfast may attenuate acute postprandial inflammation. However, when postprandial blood samples were treated with LPL, the pattern of FFA and cytokine concentrations was reversed. The FFA concentrations of LPL treated postprandial blood samples were greater than those of LPL-treated fasting blood samples . Similarly,snap clamp the cytokine concentrations from PBMCs incubated in postprandial autologous plasma treated with LPL increased compared with those from PBMC incubated with fasting plasma . The concentrations of IL-1b in postprandial whole blood treated with LPL were also increased compared with those from fasting whole blood treated with LPL . Taken together, the results from the current study and our previous mechanistic study reveal that plasma FFA concentration may be an important modulator of cytokine production in human blood. The decreased FFA concentrations in postprandial plasma compared with fasting plasma may result from the postprandial surge of insulin, which suppresses lipolysis and stimulates esterification of FFAs to TGs , leading to decreased production of cytokines. Other studies showing varying results on postprandial cytokine concentrations have not focused on the relation of the postprandial cytokine concentrations with those of plasma FFAs. Such varying results may reflect in part differences in sampling time of postprandial blood, dietary composition, and heterogeneous health status of the subject populations.
The subjects enrolled in our study were mostly young and generally healthy, with a BMI of 18–25, reflecting a relatively homogeneous group. Saturated FAs are the predominant FAs esterified in TGs of TGRLs isolated from subjects who consumed a meal high in saturated fat . Thus, the result that increased plasma concentrations of endogenous FFAs released by the treatment of LPL correlated with elevated production of cytokines in the blood samples suggests that saturated FAs in the FFA fraction predominate in modulating proinflammatory signals in blood. The breakfast meal used in this study contained 14.3 g of saturated fat, which represents 19.8% and 15.3% of the total calories from the breakfast alone and breakfast plus BB powder, respectively. Thus, the saturated fat content is more than twice the level recommended by the American Heart Association. The reason for using the elevated level of saturated fat was that, because saturated FAs can induce TLR-mediated monocyte activation, we intended to use this breakfast as a challenge meal. In spite of such a high saturated fat content of the breakfast, the postprandial plasma FFAs and cytokine concentrations declined compared with those of the fasting plasma. This result suggests that a meal-induced insulin surge may predominate in regulating plasma FFAs and, in turn, FA-induced cytokine production in healthy subjects given the type of breakfast described in our study. The surface expression of adhesion markers CD11b/c and CD14 were increased after treatment of whole blood with LPL . This result further supports the possibility that plasma FFAs derived from dietary fats can activate monocytes and may lead to increased adherence of monocytes to vascular endothelial cells. The concentrations of TGs in postprandial plasma were greatly increased compared with those of fasting plasma . FFAs, not TGs, can induce the expression of the TLR target gene product, COX-2 in macrophages . This finding is consistent with the result that the concentrations of postprandial cytokines follow FFA levels but not TG levels. Plasma FFAs are derived from TGs in adipose tissue and in part from dietary fat. However, in a microenvironment where TGRLs are exposed to LPL secreted from endothelium, local concentrations of FFAs can be increased before FFAs are taken up by endothelial cells for re-esterification to TGs or incorporation into lipid droplets . Thus, cell surface TLRs expressed in blood monocytes will be exposed to the increased FFAs and can subsequently be activated, leading to production of proinflammatory gene products including cytokines. Therefore, elevated plasma TGs could enhance the propensity of monocyte activation. Polyphenols are extensively metabolized by gut microbiota, enterocytes, and liver enzymes . Postprandial whole blood should contain any nonmetabolized polyphenols and their metabolites derived from the ingested BB powder. Therefore, to assess the effects of these absorbed polyphenols and their metabolites on TLR-mediated monocyte activation, fasting and postprandial whole blood were stimulated with endogenous FFAs derived from plasma TGs by LPL treatment in this study.