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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

2015 Annual Report

1a. Objectives (from AD-416):
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.

1b. Approach (from AD-416):
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.

3. Progress Report:
Objective 1a: Our results have demonstrated that mice without expression of a zinc transporter 7 (ZnT7 knockout where the ZnT7 gene has been removed from the DNA) become mildly insulin resistant when they reach 18 weeks of age (equivalent to 20-30 years old in humans). Both muscle and fat from Znt7 knockout mice are less sensitive to insulin resulting in increased blood sugar levels after a meal. Interestingly, insulin resistance was only observed in the fat lying under the skin, but not the fat around the internal organs of Znt7 knockout mice. Objective 1b: Our data demonstrate that ZnT7 is an important regulator of fat synthesis in fat cells. We discovered that the presence of ZnT7 in fat was differentially regulated; meaning that ZnT7 protein is only detected in mature fat cells. Without Znt7 present, fat cells are less sensitive to insulin-stimulated glucose uptake leading to a defect in fat synthesis and subsequent fat storage in the cell; a possible mechanism underlying insulin resistance observed in Znt7 knockout mice. Objective 1c: We have demonstrated that Znt7 differentially affects signaling pathways in immune cells (B cells). We confirmed that ZnT7 protein physically interacts with CD40 in B cells that activate downstream signaling pathways leading to B cell growth and production of antibodies. Objective 2. Serum samples were analyzed from 27 elderly (median age 73 ± 3 years, 47% female) Chileans with serum B-12 <120 pmol/L at baseline. These elderly were supplemented with a single intramuscular injection of 10 mg vitamin B-12. B-12 status was defined by combining B-12, total plasma homocysteine (tHcy), methylmalonic acid (MMA) and holo transcobalamin (holoTC) into one parameter (combined indicator of B-12 status, cB-12). Metabolomic response to treatment was assessed by analyzing serum before and after supplementation using targeted [166 carnitines, amino acids, sugars, glycerophospholipids and sphingolipids (BIOCRATES Absolute IDQTM)] and untargeted [247 (109 unknown) primary metabolites (GC-TOF MS)] metabolomics. Treatment increased serum B-12, holoTC and cB-12 (p<0.001), and reduced plasma tHcy and serum MMA (p<0.001). Changes pre-post treatment included increases (p<0.05) in plasmalogens, phospholipids and carnitines and reductions in one carbon metabolism moieties (i.e. methionine, cysteine and cysteine). A heat map correlation matrix revealed direct (p<0.0001) correlations between vitamin B-12 status (most strongly based on cB12) and a large family of plasmalogens, phospholipids, sphingomyelins and carnitines. Proline was inversely (p<0.0001) correlated with B-12 status. We conclude that at this time the only evidence from the metabolomics work of an effect on the Krebscycle is that changes in MMA and tHcy were associated with changes in acylcarnitines. More interesting are the strong links between cB12 and an array of plasmalogens and other phospholipids which are major constituents of the myelin sheath of nerves and myelin in the brain. There were significant, direct correlations between peripheral nerve function (less latency in the right sural nerve) of the elderly and plasmalogens and phosphatidyl carnitines. This association, revealed for the first time, could explain some of the effects of vitamin B-12 status on neurological and cognitive function. Objective 3. Examination of triglycerides, free fatty acids, and cholesterol levels in human subjects fed a zinc deficient diet suggested that zinc deficiency would increase high blood triglyceride levels while Zinc supplementation did the opposite. Metabolomic analysis of the blood samples (64) is scheduled. Objective 3. The goal of this objective was to improve and evaluate a marker of vitamin B12 status that combines the 4 available single markers, plasma vitamin B12 and total homocysteine (tHcy), methylmalonic acid (MMA) and holotranscobalamin (holoTC). The first step was to work with Dr. Sergey Fedosov of Aarhus University, Denmark, to revise his original model for the combined markers. The collaboration with Dr. Fedosov resulted in a new combined indicator which we call “combined B12” or “cB12” which can now be estimated using 2, 3 or 4 of the usual single markers. The new model can also be adjusted in the case of poor folate status. We also derived new cut-points for B12 deficiency and marginal status based on Dr. Fedosov’s modeling of data from several published studies (including our own) of the relationship between the indicator and hematology and cognitive outcomes in the elderly. We then applied our cB12 indicator to determine the effects of the pre- and post-treatment study in 51 Chilean elderly who had deficient serum B-12 (<120 pmol/L) at baseline. Response to treatment was assessed by measuring cB-12 and neurophysiology at baseline and 4 months after treatment. Treatment increased serum B-12, holoTC and cB-12 (p<0.001), and reduced plasma tHcy and serum MMA (p<0.001). Treatment produced consistent improvements in conduction in myelinated peripheral nerves; the sensory distal latency of both the left and right sural nerves improved, based on faster median conduction times of 3.1 and 3.0 msec vs. 3.3 and 3.4 msec, respectively (p<0.0001). A total of ten new sensory action potentials appeared in the two nerves after treatment. Participants with high serum folate at baseline (above median, =33.9 nmol/L) had less improvement in cB-12 (p<0.001) compared with individuals whose serum folate was below the median (<33.9 nmol/L). We conclude that asymptomatic Chilean elderly with poor B-12 status displayed improved conductivity in myelinated peripheral nerves after B-12 treatment and an interaction with folate status which was detected only by using cB-12. Additional applications of cB12 are being tested in other studies using our own data and that of collaborators, in order to further demonstrate the sensitivity and validity of this new marker of B12 status. Objective 4: Work on this project got off to a slow start due to recruitment of retirement facilities in Melbourne and the enrollment of elders into the project. It is estimated that the project is about 6 months behind schedule, but most recently University of Melbourne investigator reported that approximately 4,000 elders have now been enrolled. This is more than the original 3,000 planned, but will account for attrition and still maintain sufficient statistical power, Serum and plasma samples are received regularly. Lab analysis began in September, 2015.

4. Accomplishments
1. Zinc affects lipid synthesis in fat cells. ARS researchers at the Western Human Nutrition Research Center in Davis, California, have shown that mice in which the zinc transporter 7 (Znt7) is knocked out are mildly deficient in zinc, have low body weight gain and decreased body fat accumulation. Also, Znt7 has been shown to have differential effects on fat metabolism and insulin usage. For example, it promotes fat storage via enhanced utilization of insulin in fat lying under the skin, but not fat that surrounds internal organs in the body. Increased fat accumulation under the skin has been shown to have a protective role against insulin resistance, a preclinical phase of type 2 diabetes. Importantly, ARS researchers found that ZnT7 protein expression was induced by the fat cell maturation process which peaks in fully matured fat cells. Low ZnT7 expression affects the signaling pathways that regulate both basal and insulin-stimulated glucose uptake in fat cells, resulting in low glucose uptake and reduced fat accumulation.

2. Development and application of a new marker of vitamin B-12 status, cB12. Markers of vitamin status are important for the accurate determination of deficiencies. There are four traditionally used biomarkers of vitamin B-12 status; plasma B-12, holotranscobalamin, methylmalonic acid and total homocysteine. These markers are usually applied alone or as a ratio with one other, but this often results in an inconsistent classification of people or populations as being vitamin B-12 deficient. ARS researchers at the Western Human Nutrition Research Center, in Davis, California, in collaboration with Aarhus University, developed a new biomarker, cB12, which combines the usual four biomarkers, and have demonstrated its improved performance for detecting metabolomic and neurological responses to vitamin B-12 interventions. This research has produced a new marker of vitamin B12 status which is likely to be used widely in clinical practice, epidemiological status, and nutrition research.

Review Publications
Huang, L., Drake, V.J., Ho, E. 2015. Zinc. Advances in Nutrition. 6:224-226.

Piccolo, B., Van Loan, M.D., Gertz, E.R., Woodhouse, L.R., Souza, E., Gustafson, M.B., Campbell, C., Fiehn, O., Keim, N.L., Adams, S.H., Newman, J.W. 2015. Habitual physical activity and plasma metabolomics patterns distinguish individuals with low- versus high-weight loss during controlled energy restriction. Journal of Nutrition. 145(4):681-690. doi: 10.3945/jn.114.201574.

Dary, O., Rambelson, Z., Shahab-Ferdows, S., Dror, D., Allen, L.H., Carriquiry, A., Salman, R. 2014. Predicted efficacy of the Palestinian wheat flour fortification program: complementary analysis of biochemical and dietary data. Public Health Nutrition. 18:1950-1957.

Siddiqua, T.J., Ahmad, S.M., Ahsan, K.B., Rashid, M., Roy, A., Rahman, S.M., Shahab-Ferdows, S., Hampel, D., Ahmed, T., Allen, L.H., Raqib, R. 2015. Vitamin B12 supplementation during pregnancy and postpartum improves B12 status of both mothers and infants but vaccine response in mothers only: a randomized clinical trial in Bangladesh. European Journal of Nutrition. 54:1-13.

Fedosov, S.N., Britio, A., Miller, J.W., Green, R., Allen, L.H. 2015. Combined indicator of vitamin B12 status: modification for missing biomarkers and folate status, and recommendations for revised cut-points. Clinical Chemistry. 53(8):1215-1225.

Fialkowski, M.K., Ettienne-Gittens, R., Shvetsov, Y.B., Rivera, R.L., Van Loan, M.D., Savaiano, D.A., Boushey, C.J. 2014. Ethnicity and acculturation: do they predict weight status in a longitudinal study among Asain, Hispanic, and non-Hispanic White early adolescent females? Adolescent Health. 5:1-7.