Brief Summary of Minutes of Annual Meeting

Participants: Meeting Host - Jairam Vanamala (jairam.vanamala@colostate.edu)- Colorado State University Administrative Advisor - Tammy Bray (tammy.bray@oregonstate.edu) - Oregon State University Connie Weaver (weavercm@purdue.edu) - Purdue University Barbara Stoecker (barbara.stoecker@okstate.edu) - Oklahoma State University Barry Shane (bandie@berkeley.edu) - University of California-Berkeley Mark Failla (MFailla@ehe.osu.edu) - The Ohio State University Janos Zempleni (JZEMPLENI2@unl.edu) - University of Nebraska-Lincoln Emily Ho (emily.ho@oregonstate.edu) - Oregon State University Brian Lindshield (blindsh@k-state.edu) - Kansas State University Jennifer Teske (teskeja@email.arizona.edu) - University of Arizona Absent: Richard Bruno (Univ. of Connecticut; Edralin A. Lucas (OK State); Elvira de Mejia (U IL) Meeting was called to order at 8:30 AM. Welcome and Introductions: Participants were welcomed by the host and Colorado State University Administrators: Jairam Vanamala, Chris Melby, Department Head, Food Science and Human Nutrition; Dr. Jeff McCubbin - Dean, College of Applied Human Sciences; Craig Beyrouty, Dean, College of Agricultural Sciences. Dr. Vanamala and the administrators shared insights on value of transdisciplinary approach in addressing complex food and nutrition problems. Investigators introduced themselves and their programs. Tommy Bray spoke about brief history of W2002 and expectations for the multistate research meetings/participants. She answered questions from the audience regarding deadlines and processes for multistate renewal grant proposal. Presentations, June 4 Station reports were shared from each investigator in the following order: 1) Connie Weaver - Discussed the effects of galactooligosaccharide (GOS) on colonic calcium absorption in pre-menarcheal girls and the rapid screening method accelerator mass spectrometry for tracer quantification; Indicated that the Diet Impact on Bone is Greatest During Periods of High Bone Turnover 2) Janos Zempleni - Discussed the importance of Holocarboxylase Synthetase in Biotinylation and how grape compounds inhibits the Holocarboxylase Synthetase 3) Emily Ho - Discussed the pharmacokinetics and bioavailability of sulforaphane and its chemoprotective role in modulating epigenetic events 4) Mark Failla - Discussed oral metabolism of anthocyanins 5) Barry Shane - Discussed B-vitamins, especially B12 and folate, in relation to 1-carbon metabolism 6) Brian Lindsheild - Discussed the importance of 5alpha-reductase inhibitors in development and progression of prostate cancer 7) Jennifer Teske - Discussed the role of sleep in chronic disease development Presentations, June 5 Dr. Tammy Bray discussed the status of the W2002, its funding cycle, and indicated that the group should begin organizing for the renewal in the upcoming years. Additional station reports were shared from each investigator in the following order 1) Barbara Stoecker - Discussed ongoing nutritional studies in international populations 2) Jairam Vanamala - Discussed the in vivo anti-oxidant and anti-inflammatory activity of processed (baked vs. chipped) purple-fleshed potatoes; Presented the importance of utilizing in vitro and in vivo models in conjunction with analytical techniques in assessing the farm to fork operation on health-benefiting properties of plant foods Janos Zempleni was elected by group members to be administrative leader for the next annual cycle. Dr. Zempleni will coordinate next year's W2002 meeting in Nebraska. Objectives, timeline, and responsibilities for project renewal were discussed and decided that Dr. Zempleni, Ho and Vanamala will lead the efforts. Discussed the possibility of future collaborations among the participants and propsed to start a collaborative study on health-benefiting properties of color-fleshed potato anthocyanins.

The participants of this multi-state project have been highly productive during the past reporting period as evidenced by >50 peer-reviewed publications among attending participants and enhancement of collaborations between project members. Project objectives are listed below along with scholarly activities of the lead station. Objective 1): Determine the bioavailability (absorption, distribution, metabolism, elimination) of nutrients and other food components and their environmental and genetic determinants. Objective 2): Evaluate the bioactivity of nutrients and other food components in order to elucidate their underlying protective mechanisms. Colorado State University (Jairam Vanamala). Potato (Solanum tubersom) is the third largest source of phenolic compounds in the human diet after oranges and apples. We have shown that locally-grown purple fleshed potatoes are a greater source of bioactive compounds, even after prolonged storage and processing (baking chipping), retains in vitro anti-cancer properties, compared to white-and yellow-fleshed cultivars. These results suggest that colored potatoes are a potential healthier alternative to common potato cultivars. This year we focused on assessing anti-oxidant and anti-inflammatory properties of colored potatoes in vivo using obese pigs, an agriculturally important animal with similar nutritional metabolism to humans, as a model. Samples from 40 pigs consuming a high-fat diet for 12 weeks followed by supplementation with white vs. purple potato chips (10% vs. 20% of diet;5 weeks) were utilized to determine the effect of dose on oxidative and/or inflammatory markers. The potato diets had no effect on the food intake, weight gain and back-fat thickness. However, serum oxidative stress markers, 8-isoprostane and malondialdehyde levels, were lower in pigs consuming purple potato diet (10 %) compared to control or white potato diet. Mesenteric fat free fatty acid analysis revealed that both purple and white potato diet (10 %, Purple > White) had lower saturated fatty acids (SFA) and greater poly unsaturated fatty acids (PUFA) levels compared to the pigs consuming high-fat control. Correspondingly, serum TNF-á, a pro-inflammatory cytokine, was also suppressed by the potato diets compared to high-fat control. These results suggest that purple potatoes, even after processing, might suppress both oxidative stress/inflammatory markers in vivo via alterations in the fatty acid composition. We also completed extensive sample collection from 64 pigs (3 weeks post weaning) consuming high fat diet supplemented with (10 %) purple and white potatoes (raw, baked and chipped) for 13 weeks. An additional control (fed with low fat diet) was included to establish baseline levels of oxidative stress/inflammatory markers. University of California, Berkeley (Barry Shane). We have continued studies on the metabolic and nutritional effects of common polymorphisms in human folate-related genes that have been shown to influence disease risk. We have continued to evaluate the B12-dependent methionine synthase (MS) and methylene-tetrahydrofolate reductase (MTHFR) genetic mouse models to mimic the effects of these polymorphisms and to evaluate their effects on metabolism and how this is modified by nutritional status. We have developed a mouse model that mimics the clinical effects of human B12 and folate deficiency, and which will allow us to investigate potential adverse effects of high folate intake. We continue to evaluate genetic risk factors for neural tube defects and to identify putative modifier genes which influence folate status, homocysteine levels, and methylation potential using a number of mouse strains and a cohort of students at Trinity College, Dublin. Impact: Neural tube defects are the most common birth defects in humans and identification of genetic risk factors for this condition will allow screening to identify at risk individuals. Polymorphisms in genes encoding folate-dependent enzymes have been implicated as risk factors for cancer and vascular disease. Recently, concerns have been raised about increased cancer risk and exacerbation of B12 deficiency by folate fortification. The models we have developed may indicate whether chronic disease risk can be modified by dietary changes and may shed some insight into possible adverse effects of folate fortification. Although it has been suggested that folate fortification exacerbates vitamin B12 deficiency symptoms, our recent studies on the Trinity Student cohort do not support any adverse effects of folate fortification on vitamin B12 status in a young population. Our genetic studies may suggest novel biomarkers for assessing vitamin status. Purdue University (Connie M. Weaver). Two novel fibers, GOS and Soluble Corn Fiber, increased calcium absorption in pubertal children associated with increases in proportion of gut bifido bacteria. Equol producing capability determined through pre-screening did not affect the bone resorption attenuation response to soy consumption in postmenopausal women. Several plant extracts were effective in reducing net bone turnover in an OVX rat model. Oregon State University (Emily Ho). The classic view of cancer etiology is that genetic alterations damage DNA structure and induce mutations resulting in non-functional proteins that lead to disease progression. More recently, the role of epigenetic alterations during cancer has gained increasing attention. The lab has focused on the examination of the interaction of bioactive nutrients such as sulforaphane, catechins and zinc on mechanisms related to cancer and chronic disease development. Zinc and chronic disease: In both animal models and humans, we have found that dietary zinc deficiency increases DNA damage in peripheral blood cells. Importantly, these functional changes precede any changes in plasma zinc levels. More recently we have found that zinc status is compromised with age. Zinc supplementation in older animals reverses age-related zinc deficiency and inhibits age-related immune defects and inflammatory processes. We have also have preliminary data that suggests that zinc alters DNA methylation patterns and may be a novel mechanism by which zinc affects gene expression and the inflammatory response. Plant-derived phytochemicals from tea and cruciferous vegetables: We have found that sulforaphane, a chemical found cruciferous vegetables is an inhibitor of histone deacetylases, increases acetylated histone levels and has anti-cancer properties in the prostate. We recently reported that SFN also causes decreases in DNA methyltransferase expression and causes hypomethylation of cyclinD2, a commonly repressed and hypermethylated gene in prostate cancer cells. Other phytochemicals derived from cruciferous vegetables, such as indole-3-carbinol may also have epigenetic targets and inhibit HDAC. This work suggests that phytochemical may have the ability to alter epigenetic events that lead to disease prevention. In human supplementation trials, we have directly compared the effects of the whole food(broccoli sprouts) to commercially available supplements. We have found a significant decrease in bioavailability and impact on HDACs with supplements compared to the whole food. Surprisingly, even when supplements are pre-treated with myrosinase, the release of sulforaphane from its glucosinolate precursors in the supplement is limited. Kansas State University (Brian L. Lindsheild). We're currently conducting a study to determine the effects of providing the 5alpha-reductase inhibitors finasteride and dutasteride starting at two different times on the development and progression of prostate cancer TRAMP mice. We're also using gas chromatography to quantify and characterize the fatty acids in saw palmetto supplements. Large clinical trials have found that both finasteride and dutasteride reduced the risk of prostate cancer; however they also increased the occurrence of more advanced prostate cancer among those who did develop it. Our research will provide evidence to help clarify finasteride and dutasteride are effective and provide information on whether these should potentially be recommended for men at high risk of prostate cancer. The saw palmetto project should provide information about what supplements contain and whether they're efficacious. The Ohio State University (Mark Failla). The amount of ²-carotene delivered to the plate after processing cassava according to traditional styles of cooking and ²-carotene bioaccessibility increased in proportion to provitamin A content in transgenic high ²-carotene cultivars. ²-carotene content during storage and its bioaccessibility were greater for transgenic potato containing the OR carotenoid storage protein than that for control potato. The bioavailability of carotenoids from a vegetable salad was increased in response to the amount of co-consumed fat with a trend towards increased carotenoid absorption when the relative amount of unsaturated fat was elevated. Xanthones from a mangosteen juice were identified as the free compounds and their phase 2 metabolites in serum and urine in healthy human participants. University of Nebraska at Lincoln (Janos Zempleni). Covalent binding of biotin to histones is catalyzed by holocarboxylase synthetase (HLCS) and has been linked with the repression of genes and repeat regions in human chromatin. However, less than 0.001% of histones H3 and H4 are biotinylated, thereby raising concerns that the abundance might be too low to elicit the strong phenotypes observed in HLCS- and biotin-deficient organisms in vivo. We are proposing an alternative model that integrates the findings from previous research by us and others. In this novel model, HLCS acts as an integral part of a multiprotein gene repression complex in human chromatin, and biotinylation of histones is a mere side effect of HLCS being in close physical proximity to histones. Specifically, we are proposing that HLCS interacts physically with the following chromatin proteins that are known to mediate gene repression by creating or interpreting epigenetic gene repression marks: euchromatic H3K9 methyltransferase EHMT-1, the maintenance DNA methyltransferase DNMT1, the methyl-CpG-binding domain protein MeCP2, histone deacetylases (HDACs) 1, 2, 3, and 8, the nuclear co-repressor N-CoR, and heterochromatin protein (HP) 1. Protein-protein interactions were predicted by using a new search algorithm and verified by using co-immunoprecipitation assays, limited proteolysis assays, and yeast-two-hybrid assays. As of today, we have confirmed physical interactions between HLCS and DNMT1, MeCP2, EHMT-1, and HDAC1. Studies with N-CoR and HP1 are in progress. Studies with synthetic inhibitors and transgenic models suggest that DNA methylation is a primary loading factor, followed by docking of HLCS, followed by docking of EHMT-1 and/or HDACs. Importantly, we discovered a novel biotinylation site in EHMT-1, namely K161, which might be essential for chromatin positioning of EHMT-1 and the subsequent methylation of K9 in histone H3. Impact. Biotin and folate synergize to maintain genome stability, thereby decreasing cancer risk and the risk for birth defects. Oklahoma State University (Barbara Stoecker). A study of 41 women (over 50 years of age) with and without metabolic syndrome (MetS) indicated that in addition to differences defining MetS, mean serum adiponectin was lower in the MetS group, and circulating insulin was higher (p<0.003). Insulin resistance was significantly elevated in the MetS group as was the android:gynoid fat ratio. Android fat was positively associated with serum glucose, triacylglycerols, and insulin and with insulin resistance, whereas gynoid fat showed no significant association with these variables. Total dietary antioxidant capacity was positively correlated with serum HDL-cholesterol, adiponectin and dietary fiber intakes which supports the idea that fruits and vegetables are good sources of antioxidants. Oklahoma State University (Edralin A. Lucas). Bitter melon (Momordica charantia, MC) has been shown to improve markers of insulin resistance including adiposity, glucose and lipid profile in mice fed high fat diet. Fatty acids can activate toll-like receptor (TLR) 4 signaling pathway resulting in the enhanced production and secretion of proinflammatory cytokines which may contribute to insulin resistance associated with diet induced obesity (DIO). We examined the effect of MC on glucose and lipid homeostasis in an animal model of DIO and the role of TLR4 in mediating these effects. Eight week old male TLR4 mutant (C3H/HeJ) and control (C57BL/6) mice were randomly assigned to four dietary treatment groups for eight weeks (n=12-13/group): control (10% calories from fat), high fat (HF, 60% calories from fat), HF+1% (w/w) MC, and HF+10% (w/w) MC. Our preliminary data suggests that the C3H/HeJ strain exhibited significantly higher body weight, body fat, plasma cholesterol, and triglycerides in response to a high fat diet when compared to the C57BL/6 strain regardless of dietary treatment. However, C3H/HeJ strain had significantly lower area under the curve after a glucose tolerance test, plasma fructosamine and free fatty acid in comparison to the C57BL/6 strain. Mice fed the 10% MC, but not the 1% MC, improved glucose tolerance in the C57BL/6 but not the C3H/HeJ strain, suggesting MC may act through TLR4 signaling to modulate glucose homeostasis. The effects of MC and TLR4 mutation on mRNA expression of genes involved in glucose and lipid metabolism are being assessed. University of Connecticut (Richard Bruno). Epidemiological observations suggest that the presence of nonalcoholic fatty liver disease (NAFLD) dramatically increases the risk for cardiovascular disease (CVD). Thus, we have conducted studies to examine the extent to which phytonutrients regulate oxidative stress and inflammatory responses in experimental and clinical models of NAFLD and CVD. In a high-fat feeding model of NAFLD, we examined the extent to which green tea extract (GTE) protects against liver injury by regulating hepatic and adipose inflammatory responses under the transcriptional control of nuclear factor kappa B (NFkB). Wistar rats (16-wk old, n=63) were fed a low-fat (LF; 10% kcal) diet containing no GTE or a high-fat (HF; 60% kcal) diet containing 0, 1, or 2% GTE for 8 wks. We then examined whether GTE reduced NFkB activation and expression levels of inflammatory mediators that are regulated in a NFkB-dependent manner. In a separate clinical study, a randomized cross-over trial was conducted in healthy men to examine the extent to which gamma-tocopherol (g-T) supplementation protects against vascular dysfunction otherwise induced by acute hyperglycemia by regulating lipid peroxidation and the ratio of asymmetric dimethylarginine relative to arginine (ADMA/Arg), an index of nitric oxide bioavailability. IMPACT: The findings of our NAFLD study in rats indicated that HF feeding increased serum alanine (ALT) and aspartate aminotransferases and hepatic lipids compared to the LF group. GTE at 1 and 2% decreased ALT and hepatic lipid relative to the HF group. In liver and epididymal adipose, the HF group had lower glutathione as well as greater mRNA and protein expression of TNF-alpha and monocyte chemoattractant protein-1 (MCP-1) and NFkB binding activity than the LF group. Compared to the HF group, GTE at 2% increased glutathione and lowered protein and mRNA levels of inflammatory cytokines in both tissues. NFkB binding activities at liver and adipose were also lower, likely by inhibiting the phosphorylation of inhibitor of NFkB. NFkB binding activities in liver and adipose were correlated with ALT, and hepatic NFkB binding activity was inversely related to liver glutathione. These results suggest that GTE-mediated improvements in glutathione status are associated with the inhibition of hepatic and adipose inflammatory responses mediated by NFkB, thereby protecting against NASH. In our clinical study, healthy men completed a randomized, crossover study where they followed their usual diet or were provided g-T (500 mg/d, 5 d) prior to a fasting 75 g oral glucose challenge. Brachial artery flow-mediated dilation (FMD), plasma glucose, insulin, antioxidants, malondialdehyde (MDA), inflammatory proteins, Arg, and ADMA were measured at regular intervals during a 3 h postprandial period. g-T supplementation increased plasma g-T by 3-fold and its physiological metabolite, g-carboxyethyl-hydroxychroman by >9-fold without affecting glucose, arginine or ADMA. Baseline FMD responses, MDA, Arg, and ADMA were unaffected by g-T supplementation. Postprandial FMD decreased 30-44% following glucose ingestion, but was maintained during the g-T trial. g-T supplementation also attenuated postprandial increases in MDA that occurred following glucose ingestion. Plasma Arg decreased in both trials to a similar extent regardless of g-T supplementation. However, the ratio of ADMA/Arg increased time-dependently in both trials, but to a lesser extent following g-T supplementation. Inflammatory cytokines and cellular adhesion molecules were unaffected by glucose ingestion or g-T supplementation. Collectively, these findings suggest that short-term g-T supplementation maintained vascular function during postprandial hyperglycemia by attenuating lipid peroxidation and disruptions in nitric oxide homeostasis, independent of inflammation.

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