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ARS Home » Pacific West Area » Davis, California » Western Human Nutrition Research Center » Obesity and Metabolism Research » Research » Research Project #426640

Research Project: Novel Functions and Biomarkers for Vitamins and Minerals

Location: Obesity and Metabolism Research

2017 Annual Report

The goal of the research is to identify novel functions and biomarkers of vitamins and minerals. OBJECTIVE 1: Determine by using metabolomic approaches in animal and cell models, as appropriate, novel functions of zinc related to energy metabolism, insulin resistance in skeletal muscle, and adipose tissue and immune function. Sub-Objective 1A: Determine the mechanism of insulin resistance induced by marginal zinc deficiency in Znt7 KO mice. Sub-Objective 1B: Investigate the mechanisms underlying insulin resistance in the skeletal muscle of Znt7 KO mice. Sub-Objective 1C: Investigate the impact of mild zinc deficiency induced by the Znt7-null mutation on CD40-mediated signaling pathway activation and gene expression. OBJECTIVE 2: Discover novel functions of vitamin B12 related to energy, carbohydrate and 1-C metabolism by measuring metabolomic responses to vitamin B12 supplementation of B12 deficient humans. OBJECTIVE 3. Measure and validate novel functional biomarkers of Zn and vitamin B12 status in response to supplementation of deficient human subjects. OBJECTIVE 4. Evaluate in human intervention trials the impact of dairy consumption on measures of bone, endocrine and immune function. OBJECTIVE 5. Develop Reference Values for mineral and vitamin concentrations in human milk, which will improve estimates of recommended nutrient intakes for breastfeeding infants and their mothers.

OBJECTIVE 1: Hypothesis: Altered lipid metabolism induced by zinc insufficiency in muscle and adipose tissues contributes to glucose intolerance and insulin resistance. Advanced metabolomic, molecular and cellular technologies will be employed to determine blood and tissue signatures reflective of Zn status and pathways affected by Zn that lead to insulin resistance in muscle and mechanisms underlying Zn effects on adiposity and immune function. Tissue culture and animal models will be used, e.g., marginally Zn deficient mouse model Znt7 knockout. OBJECTIVE 2. Hypothesis: B12 supplementation of those with compromised status will alter pathways of TCA cycle, mitochondrial function, fatty acids, 1-C, amino acid and CHO metabolism. To investigate these pathways, samples from a randomized B12 supplementation trial in deficient Chilean elders will be analyzed using two metabolomic platforms. Relationships among B12 markers, metabolites and physiological functions will be evaluated before and after B12 supplementation. OBJECTIVE 3. Hypothesis: Znt7-null associated metabolite profiles of fatty acid metabolism will be used as biomarkers of zinc status. Serum collected from a Zn depletion/repletion/supplementation study will be measured for target metabolite abundance changes related to oxidative stress from zinc deficiency. Hypothesis: A combined B12 biomarker will be a better predictor of functional B12 status than single or paired biomarkers. Samples used from deficient women and their infants in Bangladesh in a randomized trial of B12 supplementation during pregnancy and lactation plus samples from Chilean elders supplemented for 18 months with B12. Responses in markers of immune function, bone turnover, and breast milk B12 will be measured, and compared to new marker of B12 status with plasma B12, homocysteine, methylmalonic acid and holotranscobalamin in Bangladeshi women. Chilean elders outcomes are neurological function, markers of inflammation, and metabolomics. The combined B12 biomarker will be evaluated in response to supplementation and associated with functional outcomes. OBJECTIVE 4. Evaluate in human intervention trials the impact of dairy consumption on measures of bone, endocrine and immune function. Hypothesis: Inclusion of 4 servings of dairy foods per day will improve bone profile, reduce fractures, improve muscle density and endocrine and inflammation profiles in elderly adults. Serum samples from a dairy interventionl in 600 ambulatory elders will be used to examine the interrelationships among bone, endocrine, immune systems and the responsiveness to dairy intake when diets with insufficient vs. adequate calcium are consumed. Objective 5. Hypothesis: Reference Values for vitamins and minerals in human milk can be established by measuring the range of concentrations in milk from well-nourished women who are not consuming additional micronutrients through supplements or fortified foods.

Progress Report
The purpose Objective 1A was to determine the mechanism by which insulin resistance was induced by marginal zinc deficiency using a mouse model (zinc transporter 7 (Znt7) mice). We discovered that a critical mediator of zinc metabolism, the Znt7, is induced as fat cells develop and accumulate lipid, suggesting Znt7 plays an important role in lipid storage in fat cells. We are extending these studies to determine how zinc deficiency alters lipid trafficking and metabolism with an emphasis on insulin resistance in skeletal muscle and liver. Insulin resistance in Znt7 knockout (KO) mice was associated with increased uptake and binding of free fatty acids in muscle cells, and fatty acid oxidative capacity in Znt7 KO muscle cells was increased as a result of enlarged mitochondria and gene expression of key enzymes involved in fatty acid beta-oxidation. As the role of mild zinc deficiency in the development of skeletal insulin resistance was previously unknown, our study has provided a molecular basis for understanding how zinc regulates lipid metabolism in skeletal muscle. Objective 1B focused on understanding the zinc mediated mechanism underlying insulin resistance in skeletal muscle. We discovered that the absence of Znt7 in the muscle cell increased fatty acid uptake into the cell leading to lipid accumulation and oxidative stress, which promoted insulin resistance. We profiled lipids and their oxidized metabolic products and found 11 out of 39 metabolites examined were increased in the Znt7 KO skeletal muscle. Among the 11 metabolites, we experimentally confirmed the negative effect of 12-hydroxyeicosatetraenoic acid (12-HETE) and 12,13-dihydroxyoctadecenoic acid (12,13-DiHOME) on the insulin signaling pathway and glucose uptake in the muscle cell. Overall, we demonstrated that increased pro-inflammatory metabolites in skeletal muscle contribute to insulin resistance. The finding that mild zinc deficiency greatly affects fatty acid uptake in skeletal muscle is novel and will greatly advance our knowledge and understanding of zinc roles in metabolic disease development. The purpose of the study in Objective 1C was to develop a biomarker for zinc status using peripheral lymphocytes. We found that cellular zinc status of immune cells affected their response to stimuli leading to decreased blood (B) lymph cell differentiation. We also found that Znt7 could influence activity of signaling pathways via regulation of cellular expression of a receptor protein on the cell surface of the B lymph cells, leading to reduced thymus (T) and B lymph cell communication. We established a new B lymph cell line and further examined Znt7 function in B lymph cells. As there are no good clinical markers for suboptimal zinc status in humans, our study will contribute to the development of a panel of reliable indicators or biomarkers to assess zinc status in humans. During the past year, we completed the first ever report of the effects of vitamin B12 supplementation on the serum metabolome in support of Objective 2. Participants were 27 elderly Chilean with B12 deficiency, but no clinical symptoms, and 18 adequate controls. We used targeted and untargeted metabolomics at baseline, and 4 months intramuscular (IM) injection with 10 milligram B12, which improved B12 status. Metabolomic changes after treatment included highly significant increases in acylcarnitines, plasmalogens and other phospholipids, while proline and other intermediates of one-carbon metabolism were reduced. There were significant correlations between nerve function with B12 status and acylcarnitines, plasmalogens, phospholipids, lyso-phospholipids and sphingomyelins. B12 status was inversely correlated with some amino acids and intermediates of energy metabolism. This research, accepted for publication, discovered new effects of B12 status on serum markers of mitochondrial function, myelin integrity, oxidative stress, and peripheral nerve function, including some implicated in Alzheimer’s and Parkinson’s diseases. Sub-objective 3A involves validating new functional biomarkers of Zn status in response to Zn supplementation of deficient human subjects. We have completed lipid and oxylipin profiling in plasma samples (128 samples in total) obtained from participants in a zinc depletion-repletion-supplementation study, in collaboration with Children’s Hospital of Oakland Research Institute, Oakland, California. We completed our statistical analysis of metabolomics and found Zn deficiency coupled with high carbohydrate intake significantly elevated blood triglycerides and arachidonic acid-derived metabolites, which are associated with oxidative stress and inflammation. Our research has clearly shown that mild zinc deficiency is implicated in development of metabolic diseases. As there are no good clinical markers for suboptimal zinc status in humans, our research will be the key to develop a panel of reliable indicators or biomarkers to assess zinc status in humans. Our focus in Sub-objective 3B has been on the further development, validation and application of our new combined biomarker of vitamin B12 status (cB12). This biomarker combines measurements of serum or plasma B12, holotranscobalamin (holoTC), methylmalonic acid (MMA) and/or total homocysteine (tHcy). The cB12 indicator has better performance than single biomarkers in several of our studies. In the trial of B12 supplementation of deficient elderly Chilean subjects (objective 2), we measured nerve conductivity. The supplement produced consistently faster conduction times in myelinated peripheral nerves and restored sensory potentials in some nerves that were undetectable at baseline. Participants with high serum folate at baseline [>33.9 nanomoles per liter (nmol/L)] had less improvement in the cB12 indicator than with lower folate. This was detected only by the cB12 indicator, producing further evidence that high folate status, which can occur when foods are fortified with folic acid, can impair B12 status. The cB12 indicator has now been used in Switzerland to show that elevated MMA and tHcy in the elderly are mainly due to impaired renal function and in the U.S., cB12 revealed that serum B12 alone has no diagnostic value in cancer patients. We are now applying cB12 to our data from an elderly Latino population in Sacramento, California and have obtained more than 7,500 data points from other investigators for modifying the cB12 model to diagnose B12 status in pregnancy and postpartum (manuscripts in preparation). We are participating in an Australian study of the effects of dairy products on bone, immunity and hormones in support of Objective 4. Due to cost of shipments from Australia we will receive samples from 300 participants rather than the planned 600. We have received 306 samples from baseline, 220 from after 3 months and 190 after 12 months of milk supplementation, which have been analyzed for cholesterol, high-density lipoprotein (HDL), low-density lipoprotein (LDL), insulin and glucose. 131 participants have a complete set of 3 matched samples and this subset will be analyzed for metabolomic outcomes. We await data from Australia to adjust our statistical analysis for covariates such as age, change in weight, body composition, and bone density. This research is partially supported by extramural funds (2032-51000-004-16T). Progress continued toward Objective 5, which involves developing Reference Values for mineral and vitamin concentrations in human milk. Although setting dietary reference intakes for lactating women, infants and young children requires data on the nutrient composition of human milk, the quantity and quality of this data is currently very poor. ARS researchers in Davis, California developed new methods of analyzing micronutrients in human milk, including liquid chromatography-mass spectrometry (LC-MS) to measure 5 B vitamins simultaneously. During the past year, with support from the Bill & Melinda Gates Foundation (2032-51000-004-19T), we wrote 7 systematic reviews on micronutrients in human milk, accepted for publication in "Advances in Nutrition." We also completed and published our study in Bangladesh on the effects of method of milk collection, time of day, and vitamin-mineral supplements on milk micronutrients, finding that supplementation had the biggest effect. In other collaborations, research was conducted on the effects of interventions on milk micronutrients; a B12 flour fortification program in Cameroon; thiamin fortified fish sauce in Cambodia; the effects of deworming and vitamin A in Peru; and lipid-based nutrient supplements in 5 countries. In November 2016, we were funded by the Bill & Melinda Gates Foundation (2032-51000-004-30T) to develop global Reference Values for nutrients in human milk. Well-nourished women and their infants will be studied for 9 months postpartum by our collaborators in Denmark, The Gambia, Brazil and Bangladesh. Since vitamin-mineral supplements affect milk nutrients, the study could not be conducted in the U.S. Breast milk volume will be measured using deuterated water. We will analyze milk macronutrients, micronutrients and oligosaccharides, milk and serum metabolomics, and the microbiome of infant fecal samples. We have: established subcontracts; finalized questionnaires; developed protocols for collecting and handling samples; formed and hosted an international Technical Advisory Group in Davis, California; and obtained Institutional Review Board approval at 2 sites. Analytical methods are mostly complete and the database is being developed in The Gambia. Recruitment will start later in 2017.

1. Zinc deficiency increases fat accumulation in muscle cells. It is known that reduced storage of fat in adipose tissue results in greater partitioning of dietary lipids to nonfat tissues, including liver and skeletal muscle, causing fat deposition, however, the underlying molecular mechanism is not completely understood. ARS researchers in Davis, California demonstrate that reduced zinc levels in skeletal muscle cells promote lipid storage in skeletal muscle via stimulation of fatty acid uptake in muscle cells. This increases lipid deposition and bioactive mediator accumulation in muscle cells resulting in inflammation, oxidative stress and insulin resistance. This is a novel discovery which demonstrates that zinc is required for maintaining fatty acid metabolism and energy usage in skeletal muscle. The finding has greatly advanced the understanding of the role and significance of zinc in lipid metabolism of the skeletal muscle and in the development of insulin resistance. It provides new insight into developing new strategies for type 2 diabetes prevention by nutritional modulations.

2. New functions of vitamin B12 are revealed by metabolomic analyses. A high proportion of the world’s population has vitamin B12 deficiency or depletion caused by their low intake of animal source foods. However, most of those people do not have clinical symptoms of B12 deficiency so it has been unclear whether or how poor B12 status affects their metabolism and function. ARS researchers in Davis, California were the first to use both metabolomic and biochemical analyses to determine the effects of supplementing vitamin B12 deficient, asymptomatic elderly with the vitamin. The B12 supplement caused large increases in acylcarnitines, plasmalogens and other phospholipids and reductions in other intermediates of one-carbon metabolism. There were significant correlations between nerve function and B12 status, acylcarnitines, plasmalogens, phospholipids, lyso-phospholipids and sphingomyelins, which suggest new mechanisms by which B12 affects neural tissue. This research discovered new effects of vitamin B12 status on human metabolism including mitochondrial function, myelin, oxidative stress, and peripheral nerve function, including some compounds previously implicated in Alzheimer’s and Parkinson’s diseases.

3. Development and validation of a new combined indicator of vitamin B12 status. Four biomarkers of vitamin B12 status are commonly used: serum or plasma B12, holotranscobalamin, methylmalonic acid, and homocysteine. Use of each marker individually, or ratios of two markers, often results in very different diagnoses of B12 status and the prevalence of inadequate status in populations. ARS scientists in Davis, California, in collaboration with Aarhus University in Denmark, have modeled thousands of biomarker values from various studies and published an algorithm that combines values of two, three or preferably all four of these markers into a new combined B12 status indicator (cB12). The ARS researchers have shown that cB12 produces better estimates of population B12 status, is correlated with nerve function in deficient elderly supplemented with B12, reveals that high folate status is associated with lower cB12, and is correlated with plasma constituents involved in nerve structure and function as well as energy metabolism. Many opportunities exist to use cB12 in clinical and population assessments of B12 status which will improve the accuracy of diagnosis, and inform policy on the prevention and treatment of B12 deficiency.

4. Improved nutrient intake recommendations for infants, young children and lactating women. It is important to know the concentration of nutrients in human milk because it is recommended as the sole source of nutrition for infants during the first six months of life, and is the basis for setting nutrient intake recommendations for infants, lactating women and young children. Unfortunately, the reported values are few and inconsistent across studies due to differences in methods and timing of milk collection, analytical methods, and maternal nutritional status. ARS researchers in Davis, California, have developed and validated more efficient methods for measuring nutrients in human milk, resulting in their leadership in a new study to obtain reference values for nutrients and other milk components in four countries. They have shown that poor maternal nutritional status and/or dietary quality results in low concentrations of many nutrients in milk and that various ways of improving micronutrient status of the mother can increase milk micronutrients. This research will improve nutrient intake recommendations for infants, lactating women and young children, and inform global public health policy about the need for maternal or infant supplementation during lactation.

Review Publications
Engle-Stone, R., Nankap, M., Ndjebayi, A.O., Allen, L.H., Shahab-Ferdows, S., Hampel, D., Killilea, D.W., Gimou, M., Houghton, L.A., Friedman, A., Tarini, A., Stamm, R.A., Brown, K.H. 2017. Iron, zinc, folate, and vitamin B12 status increased among women and children in Yaounde and Douala, Cameroon, one year after introducing fortified wheat flour. Journal of Nutrition. 147(7):1426-1436. doi: 10.3945/jn.116.245076.
Chebaya, P., Karakochuk, C.D., March, K.M., Chen, N.N., Stamm, R.A., Kroeun, H., Sophonneary, P., Borath, M., Shahab-Ferdows, S., Hampel, D., Barr, S.I., Lamers, Y., Houghton, L.A., Allen, L.H., Green, T., Whitfield, K.C. 2017. Correlations between maternal, breast milk, and infant vitamin B12 concentrations among mother-infant dyads in Vancouver, Canada and Pry Veng, Cambodia: an exploratory analysis. Nutrients. 9(3):270. doi: 10.3390/nu.9030270.
Hampel, D., Shahab-Ferdows, S., Islam, M., Peerson, J.M., Allen, L.H. 2017. Vitamin concentrations in human milk vary with time within feed, circadian rhythm, and single-dose supplementation. Journal of Nutrition. 147(4):603-611. doi: 10.3945/jn.116.242941.
Whitfield, K.C., Karakochuk, C.D., Kroeun, H., Hampel, D., Sokhonig, L., Chan, B.B., Borath, M., Sophonneary, P., Mclean, J., Talukder, A., Lynd, L.D., Li-Chan, E.C., Kitts, D.D., Allen, L.H., Green, T.J. 2016. Perinatal consumption of thiamine-fortified fish sauce in rural Cambodia. A randomized clinical trial. JAMA Pediatrics. 170(10):e162065.
Green, R., Allen, L.H., Bjorke-Monsen, A., Brito, A., Gueant, J., Miller, J.W., Molly, A.M., Nexo, E., Stabler, S., Toh, B., Ueland, P.M., Yajnik, C. 2017. Vitamin B12 deficiency. Nature Review Disease Primers.
Brito, A., Fedosov, S.N., Miller, J.W., Green, R., Uauy, R., Allen, L.H. 2016. Reply to Lawrence R. Solomon: Negative interaction of high folate status with biochemical and neurological response to vitamin B-12 treatment. American Journal of Clinical Nutrition. 103(5):1379. doi: 10.3945/ajcn.116.133322.
Townsend, M.S., Shilts, M.K., Styne, D.M., Drake, C., Lanoue, L., Woodhouse, L.R., Allen, L.H. 2016. Vegetable behavioral tool demonstrates validity with MyPlate vegetable cups and carotenoid and inflammatory biomarkers. Appetite. 107:628-638. doi: 10.1016/j.appet.2016.09.002.
Tepaamorndech, S., Oort, P.J., Kirschke, C.P., Yimeng, C., Huang, L. 2017. ZNT7 binds to CD40 and influences CD154-triggered p38 MAPK activity in B lymphocytes-a possible regulatory mechanism for zinc in immune function. FEBS Open Bio. 7(5):675–690. doi: 10.1002/2211-5463.12211.