Grant Information
| Knowledge Area | Subject of Investigation | Field of Science | Percent |
|---|---|---|---|
| 702 - Requirements and Function of Nutrients and Other Food Components | 1120 - Blueberry | 1010 - Nutrition and metabolism | 100% |
The Metabolic syndrome (MetS) is a major public health problem in the United States. The MetS is characterized by the concurrent presence of obesity, dyslipidemia, insulin resistance, glucose intolerance and hypertension, and other associated metabolic abnormalities, including endothelial dysfunction and a pro-inflammatory, pro-oxidative and pro-thrombotic environment. Finding economic and effective ways to prevent and reverse the MetS is extremely important for public health and the health care system, especially considering that its prevalence is dramatically increasing among the US population. Wild blueberries are among the commercially available fruits and vegetables that contain the highest levels of antioxidant polyphenols, mostly anthocyanins (ACNs). Past research in our laboratory has documented that wild blueberries beneficially affect the cardiovascular system and regulate vasomotor tone and also arterial metabolism in models of normal endothelial function but also dysfunction. With the proposed research, I will focus on elucidating the role of wild blueberries on endothelial dysfunction, dyslipidemia and the inflammatory state induced by Obesity as related to MetS. Through the methods mentioned above, hope to show that regular consumption of dietary achievable amounts of wild blueberries cannot only prevent the development of the abnormalities related to the MetS, but it can also significantly reduce/normalize or reverse most of the above risk factors. This can have great implications on public health, health care costs and the wild blueberry industry.
Goal: to explore the role of Wild Blueberry on Inflammation and endothelial dysfunction as related to Obesity and the Metabolic Syndrome in vivo and in vitro Animals and diets: Forty eight male OZR and their lean littermates, LZR (10-13 weeks old) will be randomly assigned to two diet groups (n=12, for each group, two sets of experiments). The diets are as follows: a Control (C, AIN93M), a Wild Blueberry-enriched diet (WB) (8% w/w substituting for dextrose in the C diet) . These levels are the equivalent of 2 cups of WB equivalent to human consumption respectively. Studies have documented beneficial effects of wild blueberries on blood lipids, memory loss and vascular function (with as low as 2% (w/w) in the diet. The animals will be fed the diets for eight weeks. Animals will be weighed weekly and food intake will be measured. They will be individually housed in stainless steel mesh-bottom cages in a room maintained at 22°C with a 12: 12 h light dark cycle. Approval is pending with the Animal Care and Use Committee of the University of Maine. Maine wild blueberries (Vaccinium angustifolium) will be freeze-dried and standardized for anthocyanin content. Future-Ceuticals (Van Drunen Farms, Momence, IL) will provide the product in vacuum-packed bags enough for each use. The product added to the animal diet, will contain 1.5% anthocyanins and will be incorporated into the C diet as described above. Diets will be prepared and stored under vacuum at -20 °C until the day before the use. Tap water and food will be provided ad libitum. The freeze-dried power will be stored in vacuum packed bags and kept in -80ºC till use. We routinely test of our wild blueberry powder at different time-periods during the year to ensure consistent anthocyanin profile during the experimental time-period. Blood and tissue collection: At the end of the experimental period, animals will be anesthetized with 95% CO2/5% O2 for two minutes. They will quickly be exanguinated by cardiac puncture and blood will be collected for immediate plasma separation, collection and storage at -80°C until subsequent analysis. The liver, part of the visceral adipose tissue, a section of the aorta and the brain will be excised, immediately snap-frozen in liquid nitrogen and stored at -80°C until further analysis for ACN concentration. The aorta (close to the aortic arch, visceral adipose and Perivascular Adipose Tissue (PVAT) tissues, will be placed in ice-cold lysis buffer (Roche Diagnostics), homogenized, sonicated for 10 seconds and centrifuged at 10,000 g for 5 minutes. The supernatant will be collected and measurement of the inflammatory markers and their gene expression thereof will be conducted. Tissue and plasma concentration of anthocyanins will be performed by means of a LC-MS/MS method that will be validated for each biological sample to be analyzed using a Quattro Micro triple quadrupole mass spectrometer (Milford, MA) equipped with an orthogonal electrospray source. Blank samples and samples spiked with different amounts of anthocyanins will be prepared and analyzed to test recovery, matrix effect, peak purity and limits of quantification, precision and repeatability. Plasma will be diluted with water, loaded onto a SPE column (HLB Oasis, Waters, Milford, MA) that has been pre-activated with methanol and washed with 2 volumes of water. The columns will be sequentially washed with 5% formic acid in water and 5% methanol in water, and then the samples will be eluted with methanol. The eluent will then be dried with nitrogen and re-suspended in 100uL with methanol:formic acid (95:5) and stored at -20ºC until analysis. Tissues will be homogenized in water:methanol:trifluoroacetic acid and centrifuged to obtain a clear solution that will be purified and concentrated by SPE procedures. Recovery of an internal standard will take into consideration sample loss during extraction procedures. The presence of metabolites such as anthocyanin glucuronides/sulphates will be verified analyzing samples before and after a treatment with glucuronidase/sulphatase enzyme. Tissue extractions will be stored at -20ºC until analysis. Blueberry anthocyanin analysis will be conducted on a BEH C18 1.7 µm (50 mm x 2.1 mm, i.d., Waters) analytical column for metabolites and a HSS C18 1.8 µm (100 mm x 2.1 mm, i.d., Waters) for anthocyanins, and the flow rate will be fixed to 400 µL/min. The eluents will be 0.05% TFA (A) and methanol:acetonitrile:water:TFA (22.5:22) Concentrations of pro-inflammatory markers in adipose, aorta and PVAT tissues: The following markers will be determined by means of commercially available rat-specific enzyme-linked immunosorbent assay (ELISA) kits, following the instructions provided by the manufacturers: TNF-α; Interleukin-6; Interleukin-8; Adiponectin (R&D Systems); VCAM-1; ICAM-1; MCP-1; P-Selectin; E-Selectin; C-Reative Protein (Uscn Life Science Inc.); i-NOS; e-NOS; PAI-1; COX-2; and NFκB (MyBioSource). Expression of pro-inflammatory markers in adipose, aorta and PVAT: mRNA from adipose tissue, aorta and PVAT will be isolated, retro-transcribed to cDNA and subjected to quantitative Real Time PCR amplification using rat-specific primer sequences as previously described (our JNB publication). In summary, mRNA from adipose, aorta and PVAT tissues will be isolated using the RNeasy Lipid Tissue Mini Kit (Qiagen, CA), while mRNA from aorta will be isolated using the RNeasy Fibrous Tissue Mini Kit (Qiagen, CA). Purity of RNA will be evaluated spectrophotometrically by determining the absorbance readings at 260 nm and 280 nm. cDNA will be synthesized from mRNA using the QuantiTect Reverse Transcription kit (Qiagen, CA), following the instructions provided by the manufacturer. The obtained cDNA will be directly used as a template for real-time PCR analysis. cDNA will then be analyzed by RT-PCR on a quantitative PCR System (Bio-Rad CFX-96) using SYBR Green Master Mix and rat-specific primer sequences targeting the genes of interest. The targeted genes will be: TNF-α; Interleukin-8; Interleukin-1; Adiponectin; VCAM-1; ICAM-1; MCP-1; P-Selectin; E-Selectin; C-reative protein; i-NOS; e-NOS; PAI-1; COX-2; VEGF and NFκB. Relative expression of the above genes will be determined by the ΔΔCt method (Livak and Schmittgen, 2001), relative to a housekeeping gene (beta-actin). Hepatocytes, adipocytes and endothelial cells will be isolated from OZR and LZR and cultured as described by Figliomeni & Abdel-Rahman (1997), Rodbell (1964) and by McGuire & Orkin respectively. Methods for studying the effect of wild blueberry diets on the regulation of the Nf-Kb pathway of inflammation on the above cell cultures are currently being set up. In brief, total and phosphorylated IκBα (Ser32/36) and IKKβ (Ser117/181) will be measured by Western blot and the expression of pro-inflammatory genes, e.g., TNF-α, IL-1β and IL-6, by quantitative realtime PCR (qPCR) will be assesed. Expected outcomes: We expect to find attenuation of pro-inflammatory markers and their gene expression in the adipose, aorta and PVAT and downregulation of theNf-Kb pathway of the OZR fed the wild blueberry diet. Limitations and pitfalls: Extensive experience in the use of animal models, including dietary intervention studies and the procedures proposed here, have been established in my laboratory in the past. Thus I do not anticipate any problems. Efforts: Presentations of results to scientific meetings, seminars at US and foreign Universities, scientific groups and through extension and outreach.
Target Audience
Science community Commodity groups Dietitians and other health professionals Consumers
Changes / Problems
Nothing Reported
Training & Professional Development
The above research project impacted graduate and undergraduate students, students conducting Honors theses and visiting scientists, not only in the area of Nutrition but also in the areas of Biochemistry and Molecular Biology and Bioengineering by acquiring skills and knowledge on berry bioactives and their effects on health as well as the Blueberry and Red Raspberry Industries and other commodity groups. Two doctoral students received their degree, two visiting scientists participated and three undergraduate students received training. Results from the above studies can be used as science-based evidence for applying for qualified health claims.
Dissemination Streams
Results from the period covered in this report were disseminated in form of a poster at the International Conference of Polyphenols and Health in Quebec, Canada (October 2017), at the WBANA Health Summit in Bar Harbor, Maine (September, 2017) in form of an oral presentation, at the Berry Health Benefits Symposium in Pismo Bay, California (March, 2017) in form of a poster, which received second price in the poster competition, and results will be presented at the American Society for Nutrition Meetings, in Boston, MA (April, 2018). Additionally, several manuscripts are in preparation to be submitted by Summer 2018. Other outputs will be presentations at invited lectures and through the media (newspaper articles, blogs and interviews). A non-provisional application to EFS for a patent to commercialize the findings related to the role of phenolics on cell migration and angiogenesis has been submitted.
Next Reporting Steps
Nothing Reported
Target Audience
Science community Commodity groups Dietitians and other health professionals General public
Changes / Problems
Nothing Reported
Training & Professional Development
Three doctoral students, 2 MS. Students, one Honor's thesis student and 2 undergraduates have been and are involved on this project.
Dissemination Streams
Yes, yearly presentations by myself and my students through invited presentations, poster presentations etc. have disseminated the results of this project nationally and internationally. For example, results have been presented yearly at the American College of Nutrition Conference, The Berry Health Benefits Symposium, The Graduate School Science Conference, University of Maine, the Wild Blueberry Health Summit, the International Conference of Polyphenols and Health, and the American Society for Nutrition. Twenty five professional presentations and invited lectures were completed during the life of the project. Nineteen abstracts were published and were presented orally or as posters.
Next Reporting Steps
Atherosclerosis is a chronic inflammatory, progressive disease of the large arteries that can lead to CVD and stroke. Angiogenesis is the formation of new capillary blood vessels from existing ones and endothelial cell migration and proliferation contribute to the development of angiogenesis; critical in the early stages of atherosclerosis. Wild blueberries (Vaccinium angustifolium) are rich in anthocyanins (ACNs) and phenolic acids (Phen) having an exceptional ranking for antioxidant capacity compared to other berries and fruits. Preliminary results on the effect of ACN and Phen fractions extracted from wild blueberries on cell migration show a differential response on endothelial cell migration based on type of fractions and fraction concentration. Studies on angiogenesis also document a concentration-depended effect, critical concentrations at which angiogenesis is modulated and differential response based on the different fractions (ACNs or Phen). ACNs seem to inhibit HUVEC migration and angiogenesis while PAs promote this process. Future experiments will determine the mechanisms behind this biological phenomenon with possible implications to atherosclerosis and wound healing <br><br>
<br>What was accomplished under these goals? Goals of the project have been completed, except for the last year's objectives. Wild blueberry bioactives have been documented to attenuate inflammation, normalize vascular function, improve dyslipidemia and regulate glucose metabolism in an animal model with the Metabolic syndrome, the Obese, Zucker rat. Our in vitro studies, with HUVECs, document that anthocyanin and phenolic extracts from wild blueberries, differentially modulate cell migration and angiogenesis in a concentration-dependent manner with implications on wound healing. <br><br><b>Publications</b><br>
Target Audience
Cardiovascular atherosclerotic disease (CAD) is a major public health problem, responsible for most deaths in developed and developing countries worldwide. Atherosclerosis is a chronic inflammatory disease of the large arteries that can result in heart disease and stroke. Early stages of atherosclerosis involve hypoxia which leads to angiogenesis mediated by endothelial cell migration and formation of new vessels. It has been documented by evaluating early human lesions that angiogenesis is a phenomenon that occurs in early atherogenesis. The American low bush Vaccinium angustifolium has an exceptional ranking for antioxidant capacity compared to other berries and fruits. Wild blueberries are rich in anthocyanins (ACNs) and phenolic acids (PA). Understanding whether and how ACNs and PAs from wild blueberries affect endothelial cell migration and angiogenesis as related to atherosclerosis is critical for public health. Through this knowledge, more effective ways may be implemented to prevent or arrest atherosclerosis and decrease the economic burden and side effects of pharmacotherapy. The above proposed research design is unique and will give a more global idea of the functions that are involved in the mechanisms by which the wild blueberries can have an effect on the cardiovascular system. This study investigates the effect of anthocyanin (ACNs) and phenolic acid (PA)-rich fractions and their combination from wild blueberry powder (Vaccinum angustifolium) on endothelial cell migration related to angiogenesis. The objectives are to study whether ACNs, PAs and their combinations affect: a. Proliferation rate of the endothelial cells and b. Speed of endothelial cell migration after acute exposure to different concentrations of ACNs, PAs and their combinations. Human umbilical vein endothelial cells (HUV-EC-C [HUVEC] (ATCC® CRL-1730™)) were used and the AlamarBlue cytotoxicity assay was performed to determine the appropriate ACN and PA concentrations for the cell migration experiments. ACNs and PA-rich fractions were extracted from freeze dried wild blueberry powder (Cherryfield, Maine, USA) and characterized by liquid chromatography (Alliance mod. 2695, Water, Milford, MA). Anthocyanins (0.0001μg/ml - 1000μg/ml) and PAs (0.0001μg/ml - 500μg/ml) were tested to determine their cytotoxicity after 24h exposure. The speed of endothelial cell migration (μm/hour) was measured by live-cell imaging (Nikon TS100) with usage of the wound healing assay dish (Ibidi, Munich, Germany). Anthocyanins at 0.002μg/ml, 8μg/ml, 15μg/ml, 60μg/ml, 120μg/ml and 300μg/ml, PAs at 0.002μg/ml, 8μg/ml, 15μg/ml, 60μg/ml, 120μg/ml and 300μg/ml and combination of both bioactive compounds at 0.002μg/ml, 8μg/ml, 15μg/ml, 60μg/ml, 120μg/ml and 300μg/ml were tested after exposure of HUVECS for a maximum of fifteen (15) Cytotoxicity assays documented that ACNs at 1000μg/ml was toxic to HUVECs and was not used in further experiments. Analysis of the time-lapse videos (TScratch, Zurich, Switzerland) documented inhibition of endothelial cell migration speed (μm/hour) when cells were treated with 60μg/ml of ACNs (28.3μm/hour), compared to control (34.3μm/hour) (p<br>
Changes / Problems
Training & Professional Development
Dissemination Streams
Next Reporting Steps
<br>What was accomplished under these goals? Under goal #1 the differential effect of anthocyanin and phenolic acid-rich extracts from wild blueberries on endothelial cell migration related to atherosclerosis were determined. Please see previous presentation. <br><br><b>Publications</b><br>
Target Audience
The Metabolic syndrome (MetS) is a major public health problem in the United States. The MetS is characterized by the concurrent presence of obesity, dyslipidemia, insulin resistance, glucose intolerance and hypertension, and other associated metabolic abnormalities. Finding economic and effective ways to prevent and reverse the MetS is extremely important for public health and the health care system, especially considering that its prevalence is dramatically increasing among the US population. Wild blueberries are among the commercially available fruits and vegetables that contain the highest levels of antioxidant polyphenols, mostly anthocyanins (ACNs). Past research in our laboratory has documented that wild blueberries beneficially affect the cardiovascular system and regulate vasomotor tone and arterial metabolism in animal models of normal endothelial function but also dysfunction, correct/normalize the dyslipidemia associated with the MetS and attenuate the inflammatory state. Preliminary results on the effects of wild blueberry consumption on plasma markers and gene expression related to glucose metabolism in the obese Zucker rat (OZR), an experimental model of metabolic syndrome and their lean littermate (LZR), documented that consumption of an 8% enriched wild blueberry diet (WB) for 8 weeks resulted in significant reduction of plasma glycated hemoglobin HbA1c, RBP4, and resistin concentrations in OZR. Additionally, following WB consumption, resistin expression was significantly down regulated in the liver of both OZR and LZR while RBP4 expression was significantly down regulated in the adipose tissue of both OZR and LZR. Thus, wild blueberry consumption normalizes some markers related to glucose metabolism and gene expression in the OZR, but has no effect on fasting blood glucose or insulin concentration. Outputs Results from the period covered in this report were disseminated at the Experimental Biology Meetings, April 2015, through published articles, presented at invited lectures and through the media (newspaper articles, blogs and interviews). Impacts Regular wild blueberry consumption normalizes markers related to glucose metabolism and gene expression that may influence the insulin resistant state associated with the Metabolic syndrome. The above research project impacted graduate, undergraduate students and visiting scientists, acquiring skills and knowledge on the area of food bioactives and their effects on health as well as the Blueberry Industry and other commodity groups.
Changes / Problems
Nothing Reported
Training & Professional Development
Graduate, undergraduate students and visiting scientists aquiring skills and knowledge on the area of food bioactives and their effects on health as well as the Blueberry industry and other commodity groups. PARTICIPANTS: Stefano Vendrame and Panagiotis Tsakiroglou, doctoral candidates, and Thomas Merrow and Vasiliki Papakotsi, Masters students and Christianna Dagher, undergraduate student at the Department of Food Science and Human Nutrition, School of Food and Agriculture University of Maine.
Dissemination Streams
Through published articles, professional presentations and invited presentations to community groups, and through media (newspaper articles, blogs and interviews).
Next Reporting Steps
During the third year of this project, the role of wild blueberrieson the endothelial dysfunction associated with Obesity and the Metabolic Syndrome will be explored. More specifically, the role of perivascular adipose tissue (PVAT) and its contribution to vascular function in Obesity and the role of wild blueberry consumption on modulating PVAT in relationship to Obesity will be assessed. <br><br>
<br>What was accomplished under these goals? Impaired fasting blood glucose is one of the landmark signs of metabolic syndrome, together with hyperinsulinemia, dyslipidemia, hypertension, and a chronic pro-inflammatory, pro-oxidative and pro-thrombotic environment. This study investigates the effect of wild blueberry consumption on blood glucose levels and other parameters involved in glucose metabolism in the obese Zucker rat (OZR), an experimental model of metabolic syndrome. Sixteen OZR and 16 lean littermate controls (LZR) were fed an 8% enriched wild blueberry diet (WB) or a control diet (C) for 8 weeks. Plasma concentrations of glucose, insulin, glycated hemoglobin GHbA1c, resistin and retinol binding protein 4 (RBP4) were measured. Expression of the resistin, RBP4 and glucose transporter GLUT4 genes were also determined, both in the liver and the abdominal adipose tissue (AAT). Plasma glycated hemoglobin HbA1c, RBP4, and resistin concentrations were significantly lower in OZR following the WB diet (-20%, -22% and -27% respectively, compared to C diet, p<br><b>Publications</b><br>
Target Audience
Science Community Commodity groups Dietitians and other health care professionals General public
Changes / Problems
Nothing Reported
Training & Professional Development
Graduate, undergraduate students and visiting scientists aquiring skills and knowledge on the area of food bioactives and their effects on health as well as the Blueberry industry and other commodity groups. PARTICIPANTS: Stefano Vendrame and Panagiotis Tsakiroglou, doctoral candidates, Department of Food Science and Human Nutrition, University of Maine. Thomas Merrow and Vasiliki Papakotsi undergraduate students, Department of Food Science and Human Nutrition,University of Maine.
Dissemination Streams
Through published articles, professional presentations and invited presentations to community groups, and through media (newspaper articles, blogs and interviews). See underoutputs section.
Next Reporting Steps
During the second year of this project, the role of wild blueberries on the endothelial dysfunction associated with Obesity and the Metabolic Syndrome will be explored. Additionally, the role of perivascular adipose tissue (PVAT) and its contribution to vascular function in Obesity will be studied and the role of wild blueberry consumption on modulating PVAT in relation to Obesity will be assessed. <br><br>
<br>What was accomplished under these goals? Goal: to explore the role of Wild Blueberry on Inflammation as related to Obesity and the Metabolic Syndrome in vivo and in vitro. Objectives:This study investigates the ability of a wild blueberry-enriched diet to improve the proinflammatory status associated with the Metabolic Syndrome in the obese Zucker rat (OZR) by studying circulating levels of pro- and anti inflammatory markers and their gene expression in the liver and adipose tissues. Animals and diets: Twenty male OZR and twenty of their lean littermates, LZR (10-13 weeks old) were randomly assigned to two diet groups (n=10, for each group). The dietswere as follows: a Control (C, AIN93M), a Wild Blueberry-enriched diet (WB) (8% w/w substituting for dextrose in the C diet). The animals were fed the diets for eight weeeks, weighed weekly and food intake was measured. Maine wild blueberries (Vaccinium angustifolium) were freeze-dried and standardized for anthocyanin content. Future-Ceuticals (Van Drunen Farms, Momence, IL) provided the product in vacuum-packed bags enough for each use. The product added to the animal diet,contained 1.5% anthocyanins. Diets wereprepared and stored under vacuum at -20 °C until the day before the use. Tap water and food were provided ad libitum. Blood and tissue collection: At the end of the experimental period, animals were anesthetized with 95% CO2/5% O2 for two minutes. They were exanguinated by cardiac puncture and blood was collected for immediate plasma separation, collection and storage at -80°C until subsequent analysis. The liver and the visceral adipose tissue wereexcised and were placed in ice-cold lysis buffer (Roche Diagnostics), homogenized, sonicated for 10 seconds and centrifuged at 10,000 g for 5 minutes. The supernatant was collected and measurement of the inflammatory markers and their gene expression was conducted. Circulating Markers of Inflammation. Plasma samples were analyzed for four markers of inflammation by means of commercially available immunoassay kits. Tumor Necrosis Factor Alpha (TNF-α) was determined using the Quantikine Rat TNF-α Immunoassay kit (R&D Systems #RTA00); interleukin 6 (IL-6) was measured using the Quantikine Rat IL-6 Immunoassay kit (R&D Systems #R6000B); adiponectin was measured using the Rat Adiponectin ELISA Kit (Millipore #EZRADP-62K); C-reactive protein (CRP) was measured using the high-sensitivity Rat CRP Elisa Kit (Millipore #CYT294). Expression of CRP, IL-6, TNF-α, adiponectin and Nf-kB in liver and adipose tissue. Briefly, mRNA from liver and abdominal adipose tissues was isolated, retro-transcribed to cDNA and subjected to two steps, real time, reverse transcription PCR amplification using rat-specific primer sequences for the CRP, IL-6, TNF-α, adiponectin and Nf-kB genes. mRNA from frozen fat fragments was isolated using the RNeasy Lipid Tissue Mini Kit (Qiagen #74804), while mRNA from liver was isolated using the RNeasy Mini Kit (Qiagen #74104). Quality and quantity of extracted mRNA was determined spectrophotometrically, measuring absorbance at 260 nm and 280 nm wavelengths in UV transparent cuvettes. Reverse transcription to cDNA and genomic DNA elimination was performed using the QuantiTect Reverse Transcription Kit (Qiagen #205313).The reverse transcription product was subjected to RT-PCR on a quantitative PCR System (Bio-Rad CFX96) using Sybr Green master mix (SSoFast EvaGreen, Bio Rad #172-5202) and rat-specific primer sequences targeting the genes of interest. For each primer and tissue (liver or adipose), the analysis was performed in triplicate with a reaction volume of 20 µL per well (1.5 µL reverse transcription product, 10 µL Sybr Green Mix, 2 µL primers forward + reverse, and 6.5 µL RNAse free water). After an enzyme activation step (95°C x 30s), 45 amplification cycles were performed (denaturation at 95°C x 2s, annealing/extension at 60°C for 5s) followed by a melting curve (75-95°C in 0.5°C increases, 2s per step) to ensure specificity of amplification. Relative expression of the genes of interest was determined by the ΔΔCt method as described by Livak and Schmittgen (Livack and Scmittgen, 2001), relative to a housekeeping gene (beta-actin) and expressed as fold-variation following WB treatment compared to the control animals. In OZR, WB consumption resulted in decreased plasma concentrations of TNF-α (-25.6%, P<br><b>Publications</b><br>