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 Highbush Blueberry Council and was composed of a 50/50 mixture of 2 varieties of highbush 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.EDTA-treated plasma was collected at fasting and postprandial blood draws and concentrations of plasma cytokines were determined by using the Human ProInflammatory II 4-plex ultra-sensitive kit from Meso Scale Discovery. Plasma glucose and serum TGs, total cholesterol, growing blueberries in pots and HDL cholesterol were measured by using standard clinical chemistry techniques on a COBAS Integra 400 plus analyzer according to the manufacturer s instructions . Serum FFAs were measured by using NEFA-HR2 reagents on the COBAS Integra 400 plus.
LDL cholesterol was calculated by using the Friedewald equation . Serum insulin was measured by using an Immulite analyzer .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 monocytespecific 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 LPLtreated postprandial blood samples were greater than those of LPL-treated fasting blood samples . Similarly, 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. Certain polyphenol compounds found in fruits and vegetables can inhibit the activation of pattern recognition receptors . BBs contain high concentrations of these polyphenolic compounds including anthocyanins . Cyanidin-3-glucoside and their metabolites suppress LPS-induced cytokine production in THP-1 monocytes . BB supplementation did not affect postprandial FFA and cytokine concentrations ; however, drainage gutter it attenuated the propensity of monocyte activation induced by elevated concentrations of FFAs in LPL-treated blood ex vivo . Supplementation with 2 and 4 servings of BBs with the breakfast meal resulted in decreased LPL-induced secretion of IL-1b and IL-6, respectively, in postprandial whole blood without affecting concentrations of FFAs released . These results suggest that the consumption of BB powder with the breakfast meal could decrease the propensity of postprandial monocyte activation in the microenvironment where blood monocytes directly interact with endothelial cells that secrete LPL.
The inhibitory effect of the BB powder on LPL induced cytokine production is likely due to the inhibitory effects of polyphenols on FFA-induced activation of pattern recognition receptors including TLRs rather than potential offtarget effects on LPL because BB intake did not alter the concentrations of FFAs released. It was reported that intake of strawberries , orange juice , tomatoes , or blackcurrants with a high-fat meal was cardioprotective by decreasing selective inflammatory markers . Because postprandial FFA concentrations may change in time, the impact of polyphenol-rich BBs on FFA-induced postprandial inflammatory status will need to be studied in the future with blood samples taken at multiple time points, including the period when FFA concentrations rebound close to or exceed fasting concentrations. The study has several limitations. The study was not powered to look at interactions between sex and consumption of BB powder. In addition, the subjects were allowed to return to their normal daily activities after consumption of the test meal until their return for the postprandial blood draw. Because sex differences and physical activity could have affected postprandial responses, recruitment strategies to create a balanced sex distribution and control of physical activity may need to be implemented in future studies. In summary, consumption of an MHF breakfast decreased postprandial plasma FFA and cytokine concentrations compared with those of fasting plasma, suggesting that eating breakfast acutely attenuates the inflammatory status in postprandial blood. Plasma FFA concentrations may be an important determinant modulating monocyte activation as assessed by TLR-mediated IL-1b secretion and the expression of adhesion molecules . These results corroborate the results from our previous mechanistic studies that both palmitic acid and endogenous FFAs can directly activate TLR2 and induce the expression of proin- flammatory cytokines in primary monocytes and whole blood. A corollary of these results is that the concentration of plasma FFAs may be one of the important determinants affecting the inflammatory status in blood. Thus, our results lead to the next translational question as to whether plasma FFA concentrations can be a target of dietary or pharmacological intervention to alleviate the increased inflammation in metabolic diseases. Supplementation with BB powder did not affect the postprandial FFA and cytokine concentrations; however, it suppressed FFA-induced cytokine production in LPL-treated blood.Grapevine berry ripening can be divided into three major stages. In stage 1, berry size increases sigmoidally. Stage 2 is known as a lag phase where there is no increase in berry size. Stage 3 is considered the ripening stage. Veraison is at the beginning of the ripening stage and is characterized by the initiation of color development, softening of the berry and rapid accumulation of the hexoses, glucose and fructose. Berry growth is sigmoidal in Stage 3 and the berries double in size. Many of the flavor compounds and volatile aromas are derived from the skin and synthesized at the end of this stage. Many grape flavor compounds are produced as glycosylated, cysteinylated and glutathionylated precursors and phenolics and many of the precursors of the flavor compounds are converted to various flavors by yeast during the fermentation process of wine. Nevertheless, there are distinct fruit flavors and aromas that are produced and can be tasted in the fruit, many of which are derived from terpenoids, fatty acids and amino acids. Terpenes are important compounds for distinguishing important cultivar fruit characteristics. There are 69 putatively functional, 20 partial and 63 partial pseudogenes in the terpene synthase family that have been identified in the Pinot Noir reference genome. Terpene synthases are multi-functional enzymes using multiple substrates and producing multiple products. More than half of the putatively functional terpene synthases in the Pinot Noir reference genome have been functionally annotated experimentally and distinct differences have been found in some of these enzymes amongst three grape varieties: Pinot Noir, Cabernet Sauvignon and Gewürztraminer. Other aromatic compounds also contribute significant cultivar characteristics. C13-norisoprenoids are flavor compounds derived from carotenoids by the action of the carotenoid cleavage dioxygenase enzymes.