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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

2016 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 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.

Progress Report
Objective 1A. We found that Znt7 (zinc transporter 7) knockout mice (without the protein expressed in the body) became insulin resistant mainly due to reduced activity of Akt in insulin sensitive fat tissue. Decreased Akt activity in fat tissue results in reduced lipid synthesis and leads to metabolic redistribution of fatty acids from fat to skeletal muscle. Objective 1B. We found that reduced glucose uptake due to attenuated Akt activation as well as increased accumulation of long chain fatty acids were the causative reasons for the severe insulin resistance and impaired glucose metabolism observed in Znt7 knockout mice fed a high carbohydrate diet. Objective 1C. We demonstrated that the activation of p38 MAPK was negatively affected by cellular zinc depletion in B lymphocytes while zinc supplementation positively influenced the activity of p38 MAPK. We showed that reduction in Znt7 expression in B lymphocytes reduced cell surface expression of CD40. We also showed that reduction of ZnT7 expression in B lymphocytes decreased TNFa mRNA expression while increased ZnT7 expression in the cell augmented TNFa mRNA expression. Objective 2. This year we continued work on the interpretation of metabolomic analysis of the effects of vitamin B-12 supplementation in a group of Chilean elderly with deficient and marginal vitamin B-12 status at baseline. In the pre-post treatment study we gave a single intramuscular injection of 10 mg vitamin B-12 to 27 community-dwelling Chileans (median age 73 ± 3 years 63% female) with confirmed low vitamin B-12 status evaluated with at least three out of four biomarkers: serum vitamin B-12, and plasma total homocysteine (tHcy), methylmalonic acid (MMA) and holotranscobalamin (holoTC). A new “combined” indicator of vitamin B-12 status (cB-12) was computed by combining these biomarkers. Targeted metabolites were measured at baseline and four months after treatment. Using matrix correlation analyses, connections between metabolomic variables and B-12 status were explored. Data were also included from a group of elderly participants with adequate vitamin B-12 status (n=18) to expand the range of status explored with targeted metabolomics. The single injection increased serum vitamin B-12, holoTC and cB-12 (p<0.001), and reduced plasma tHcy and serum MMA (p<0.001). Changes from pre- to post-treatment included increases (p<0.001) in acylcarnitines, plasmalogens and other phospholipids while proline and intermediaries of one-carbon metabolism, i.e. methionine and cysteine, were reduced (p<0.001). In the overall group, including those with adequate B-12 status, there were significant positive correlations (p<0.05) between acylcarnitines, plasmalogens, phospholipids, lyso-phospholipids and sphingomyelins versus vitamin B-12 status in the matrix correlation analyses. There were positive and negative relationships (p<0.05) between vitamin B-12 status and the primary metabolites. This is the first characterization of the human serum metabolome in sub-clinical vitamin B-12 deficiency, and its response to supplementation. Metabolomics revealed connections between sub-clinical vitamin B-12 deficiency and serum metabolic markers of mitochondrial function, myelin integrity and peripheral nerve function, some factors previously implicated in Alzheimer’s and Parkinson’s diseases. Objective 3A. We found in our animal study that zinc deficiency, but not a zinc adequate/supplemented condition, had specific effects on lipid metabolism. We wanted to confirm our findings in blood samples collected from human study participants (in collaboration with researchers at the Children’s Hospital Oakland Research Institute). We have completed measurements on samples collected from 18 human subjects for plasma triglycerides, free fatty acids, and total cholesterols. We have also completed fatty acid composition analysis and oxylipin determination using these plasma samples. We found that zinc deficiency increased blood triglyceride levels. Blood cholesterol and free fatty acid levels were not affected by zinc deficiency in human subjects. Objective 3B. During 2015 we developed and published a new “combined” marker of vitamin B-12 status (c-B12) by combining traditional, individual markers of vitamin B12 status: serum vitamin B-12 and holotranscobalamin, plasma total homocysteine (tHcy), and methylmalonic acid (MMA). While combining the four measures of status is ideal, we also developed a way to calculate c-B12 from two or three of these markers. During this year we tested the ability of c-B12 to detect the effects of treating vitamin B-12 deficient elderly, who had no clinical symptoms of deficiency, on their ability to conduct impulses in peripheral nerves. It is uncertain whether vitamin B-12 supplementation can improve neurophysiologic function in asymptomatic elderly with low vitamin B-12 status or whether high folate status adversely affects neurological responses to vitamin B-12 supplementation. We assessed the effects of a single intramuscular injection of 10 mg vitamin B-12 (which also contained 100 mg vitamin B-6 and 100 mg vitamin B-1) on vitamin B-12 status and neurophysiologic function (speed of impulse conduction in peripheral nerves) in elderly community-dwelling Chileans with low serum vitamin B-12 concentrations who were consuming bread fortified with relatively high levels of folic acid. The 51 participants (median age: 73 years; females: 47%) had serum vitamin B-12 concentrations indicating deficiency i.e., <120 pmol/L, at screening. Vitamin B-12 status was defined by measuring serum B-12, plasma total homocysteine (tHcy), methylmalonic acid (MMA), and holotranscobalamin and combining them to calculate the single combined indicator of vitamin B-12 status (cB-12). cB-12 and neurophysiologic variables were measured at baseline and four months after treatment. Treatment with vitamin B-12 increased serum vitamin B-12, holotranscobalamin, and cB-12 and reduced plasma tHcy and serum MMA. Treatment also improved impulse conduction in myelinated peripheral nerves; the sensory latency of both the left and right sural nerves improved as evidenced by faster conduction of stimuli in the nerves. In addition, in 10 participants, responses were observed in sural nerves after treatment which were not detectable before treatment. Participants with high serum folate at baseline (>33.9 nmol/L) had less improvement in cB-12 than did individuals whose serum folate was less than the median concentration. We conclude that asymptomatic Chilean elderly with poor vitamin B-12 status had improved conductivity in myelinated peripheral nerves after vitamin B-12 treatment. However, those with higher serum folate concentrations had less improvement in nerve function. This interaction with folate status was detected only with the use of cB-12, and was not detectable using traditional single markers of vitamin B-12 status. Objective 4. In conjunction with colleagues in Melbourne Australia, ARS researchers are evaluating the impact of four servings/day of dairy versus the standard two servings/day on markers of cardiovascular and immune health in elderly men and women in retirement facilities. Recruitment of retirement facilities in Melbourne has been completed and 4,000 residents enrolled in the project. To date, serum samples have been collected from 290 individuals including baseline, three month and 12 month samples. Samples have been cross-coded for matching ID numbers, birth dates, sex, and facility. To date there are 203 baseline samples from females and 86 baseline samples from males. Additionally, there are 117 and 60 3 month samples for females and males, respectively. At the 12 month mark there are samples for 62 females and 25 males. Analytical laboratory analysis for inflammatory markers has been completed on a subset of women and men that have completed 12 months of intervention. Completion of all participants is expected in late 2017.

1. Weight loss impacts bone loss and health. Contrary to popular opinion, weight loss can be detrimental to bone health because there is a reduction in calcium and vitamin D intake as well as a major change in the mechanical load applied to the skeleton. To further understand the impact of weight loss on bone health, researchers in Davis, California examined changes in bone turnover markers, endocrine and inflammatory outcomes in a group of obese individuals after Roux-En-Y gastric bypass surgery. Twenty women and men were followed before and 12 months after surgery. Before surgery fasting, glucose and fatty acids accounted for 35% of the variability in the bone formation-to-breakdown ratio; one month post-surgery, the formation-to-breakdown ratio declined significantly and remained low for the following 11 months. Interestingly the change in body weight or composition did not explain the decline in the formation-to-breakdown ratio thus change in mechanical load does not appear to be a major contributing factor to bone loss. ARS researchers did find that blood glucose and insulin, plus acute inflammatory marker C-reactive protein, explained 48% of the variation in the formation-to-breakdown ratio.

2. Milk fat globule membrane (MFGM) attenuates impact of higher saturated fat intake. The U.S. Dietary Guideline to limit saturated fat intake is based on epidemiological data that showed high saturated fat intake is associated with cardiovascular disease. However, results from human research studies suggest otherwise, so ARS researchers in Davis, California examined the impact of a high saturated fat meal on inflammatory markers in obese men and women for six hours following the meal. Two different forms of saturated fat (palm oil and whipping cream) were ingested with and without the addition of milk fat globule membrane (MFGM). MFGM is found in dairy products and surrounding the fat globules in milk and has been shown to reduce the inflammatory response to food ingestion. ARS researchers found that consumption of MFGM with either palm oil or whipping cream resulted in lower total cholesterol, LDL-cholesterol, insulin and the inflammatory marker soluble adhesion molecule. The addition of MFGM attenuates the negative impact of a high saturated fat meal in overweight and obese men and women.

3. Zinc deficiency impacts lipid metabolism. Zinc status affects insulin-stimulated blood glucose uptake into fat and muscle and fat deposition in fat tissue, but the underlying mechanisms are not understood. ARS researchers in Davis, California found that decreased Akt activation (an enzyme that promotes glucose uptake into cells) and lipid formation in fat tissue underlies severe insulin resistance and impaired glucose metabolism in Znt7 knockout animals (no Znt7 expression in the body), a model for mild zinc deficiency in humans. Additionally, ARS researchers found that altered fatty acid redistribution from fat to muscle is the major event that causes the severe insulin resistance and impaired glucose metabolism in Znt7 KO mice fed a high carbohydrate diet. The study indicates that zinc deficiency has adverse effects on fat metabolism. A high carbohydrate diet worsens this effect in both Znt7 knockout mice and humans.

4. Zinc deficiency impairs immune function. Zinc deficiency causes impaired immune function leading to increased risk of infection, such as upper respiratory infection and diarrhea. However, the underlying molecular mechanism is not completely understood. ARS researchers demonstrated that the activation of CD40 ligand-induced p38 MAPK is negatively affected by cellular zinc chelation in B lymphocytes while zinc supplementation positively influences the activity of p38 MAPK. They also demonstrated that reduction in Znt7 expression in B lymphocytes reduces cell surface expression of CD40, which leads to reduced B lymphocyte immune response to T cell-mediated B cell activation. This novel discovery points out that the CD40 signaling pathway is regulated by zinc. The study provides fundamental knowledge for identification of biomarkers of zinc deficiency using blood lymphocytes collected in clinic and field settings.

5. Role of zinc transporter ZnT8 in development of diabetes. Haploinsufficiency of ZnT8 (one good allele and one mutated allele) in humans reduces the risk of type 2 diabetes (T2D). How the haploinsufficiency of ZnT8 protects individuals from T2D is currently not understood. ARS researchers found that the protective effect of allelic ZnT8 deficiency on T2D development is likely via an Akt-dependent beta-cell survival mechanism. These findings have greatly advanced the understanding of the role and significance of ZnT8 in the development of diabetes and provide new insight into developing new strategies for T2D prevention and/or treatment.

6. Vitamin B-12 supplementation improves nerve function in deficient elderly. A high proportion of the world’s population has poor vitamin B-12 status, mainly because they do not consume an adequate amount of animal source foods, which are the only food source of the vitamin. However, the adverse metabolic effects of poor vitamin B-12 status in such populations are usually not clear because severe clinical symptoms do not usually occur. ARS researchers in Davis, California evaluated the metabolic effects of supplementing elderly people with poor vitamin B-12 status with a high dose of the vitamin. This approach used targeted metabolomics (using a kit that measured 181 serum metabolites with mass spectrometry) before and after supplementation, in the first characterization of the human serum metabolome in sub-clinical vitamin B-12 deficiency and its changes in response to supplementation. ARS researchers found connections between sub-clinical vitamin B-12 deficiency status and serum metabolic markers of mitochondrial function, myelin integrity and peripheral nerve function, some factors previously implicated in Alzheimer’s and Parkinson’s diseases. These results provide new markers of vitamin B-12 status, and suggest the need for trials that provide supplemental vitamin B-12 to patients with Alzheimer’s, Parkinson’s and other neurological diseases.

7. A new vitamin B-12 marker distinguishes neural impairment in B-12 deficiency and effects of high folate status. Although it is well-established that severe vitamin B-12 deficiency results in impaired nerve function and clinical signs such as neuropathy (numbness and tingling in arms and legs), it has been more difficult to detect signs of poor nerve function in less severe deficiency. ARS researchers in Davis, California approached this problem by evaluating vitamin B-12 status with a new indicator (c-B12) that combines four traditional status markers, and measuring nerve conductance in the arms and legs of vitamin B-12 deficient Chilean elderly before and after they received a supplemental dose of the vitamin. These elderly had improved conductivity in myelinated peripheral nerves after vitamin B-12 treatment. However, those with higher serum folate concentrations, as a result of a folic acid flour fortification program, had less improvement in nerve function. This interaction with folate status was detected only with the use of cB-12, and was not detectable using traditional single markers of vitamin B-12 status. The research validates the usefulness of the new marker of vitamin B-12 status, shows that neurological function is indeed impaired in B-12 deficiency even when there are no overt clinical symptoms of deficiency, and suggests that high levels of folic acid addition to flour in fortification programs may be adversely affecting nerve function.


Review Publications
Demmer, E., Van Loan, M.D., Rivera, N., Rogers, T.S., Gertz, E.R., German, J., Smilowitz, J.T., Zivkovic, A.M. 2016. Addition of a dairy rich milk fat globule membrane to a high-saturated fat meal reduces the postprandial insulinaemic and inflammatory response in overweight and obese adults. Journal of Nutritional Science. 5(e14):1-11. doi: 10.1017/jns.2015.42.
Tepaamorndech, S., Kirschke, C.P., Pedersen, T.L., Keyes, W.R., Newman, J.W., Huang, L. 2015. Zinc transporter 7 deficiency affects lipid synthesis in adipocytes by inhibiting insulin-dependent Akt activity and glucose uptake. FEBS Journal. 283(2):378-394. doi: 10.1111/febs.13582.
Demmer, E., Van Loan, M.D., Rivera, N., Rogers, T.S., Gertz, E.R., German, J.B., Zivkovic, A.M., Smilowitz, J.T. 2016. Consumption of high-fat meal containing cheese compared to a vegan alternative lowers postprandial C-reactive protein in overweight and obese individuals with metabolic abnormalities: a randomised controlled crossover study. Nutritional Science. 5(e9):1-12. doi: 10.1017/jns.2015.40.
Allen, L.H. 2016. Current information gaps in micronutrient research, programs and policy: how can we fill them? World Review of Nutrition and Dietetics. 115:109-117.
Rogers, T.S., Demmer, E., Richardson, C., Gertz, E.R., Buchholz, B., Hillegonds, D., Garrod, M.G., Van Loan, M.D. 2016. Is bone equally responsive to calcium and vitamins D intake from food vs. supplements? Use of 41Ca tracer kinetic model. Bone Reports. 5:117-123. doi: 10.1016/j.bonr.2016.05.001.
Rogers, T.S., Swarbrick, M.M., Wolfe, B.M., Ali, M.R., Blankenship, J., Stanhope, K.L., Havel, P.J., Van Loan, M.D. 2015. Effects of endocrine and inflammatory changes on markers of bone turnover following Roux-en-Y gastric bypass surgery. Medical Science Research. doi: 10.18103/mra.v2i2.329.
Huang, L., Kirschke, C.P. 2016. Down-regulation of zinc transporter 8 (SLC30A8) in pancreatic beta-cells promotes cell survival. Austin Journal of Endocrinology and Diabetes. 3:1037.
Hampel, D., Allen, L.H. 2016. Analyzing B-vitamins in human milk: methodological approaches. Critical Reviews in Food Science and Nutrition. 56:494-511.
Williams, A.M., Chantry, C.J., Young, S.L., Allen, L.H., Arnold, B.F., Colford Jr, J.M., Dentz, H.N., Hampel, D., Lin, A., Null, C.A., Shahab-Ferdows, S., Stewart, C. 2016. Vitamin B-12 concentrations in breast milk are low and are not associated with reported household hunger, recent animal source food or vitamin B-12 intake among women in rural Kenya. Journal of Nutrition. 146(5)1125-1131. doi: 10.3945/jn.115.228189.
Hampel, D., Shahab-Ferdows, S., Adair, L.S., Bentley, M.E., Flax, V.L., Jamieson, D.J., Ellington, S.R., Tegha, G., Chasela, C.S., Kamwendo, D., Allen, L.H. 2016. Thiamin and riboflavin vitamers in human milk: effects of lipid-based nutrient supplementation and stage of lactation on vitamer secretion and contributions to total vitamin content. PLoS One. doi: 10.1371/journal.pone.0149479.
Whitfield, K.C., Karakochuk, C.D., Kroeun, H., Hampel, D., Sokhoing, L., Chan, B.B., Borath, M., Sophonneary, P., Mclean, J., Aminuzzaman, T., Lynd, L.D., Li-Chan, E.Y., Kitts, D.D., Green, T.J. 2016. Perinatal consumption of thiamin-fortified fish sauce in rural Cambodia: a randomized controlled efficacy trial. JAMA Pediatrics. doi: 10.1001/jamapediatrics\2016.2065.
Brito, A., Miller, J.W., Green, R., Fedosov, S.N., Harvey, D., Shahab-Ferdows, S., Verdugo, R., Sanchez, H., Albala, C., Uauay, R., Allen, L.H. 2016. Vitamin B-12 treatment of asymptomatic, deficient, elderly Chileans improves conductivity in myelinated periphreal nerves, but high serum folate impairs vitamin B-12 status response assessed by the combined indicator of vitamin B-12 status. American Journal of Clinical Nutrition. 103:250-257. doi: 10.3945/ajcn.115.116509.