Location: Obesity and Metabolism Research
2019 Annual Report
Objectives
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.
Approach
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
This is the final report for project 2032-51000-004-00D, which expired January 2019 and continues research in project 2032-51000-005-00D.
Objective 1 was to determine by using metabolomic approaches in animal and cell models, as appropriate, novel functions of zinc (Zn) related to energy metabolism, insulin resistance in skeletal muscle, and adipose tissue and immune function. ARS researchers in Davis, California, discovered that zinc transporter 7 (ZnT7) deficiency via knockout (KO) in mice induced insulin resistance when mice reached 16-18 weeks of age. Both skeletal muscle and adipose tissues from Znt7 KO mice had reduced Akt activation, a key molecule and event during insulin-stimulated glucose uptake in muscle and fat after a meal. The amount of ZnT7 protein in fat cells was coordinated with fat cell development. Reduction in ZnT7 protein expression had a significantly negative impact on fat storage in fat tissue leading to metabolic redistribution of FAs from fat tissue to skeletal muscle. Accumulation of fats and long-chain FAs in skeletal muscle was a cause of severe insulin resistance and impaired glucose metabolism observed in ZnT7 knockout mice fed a high fat or high carbohydrate diet. Another important discovery was the target molecules regulated by Zn in immune cells. Using an in vitro system, the ARS researchers demonstrated that Zn was a regulator of the CD154-CD40-mediated action of downstream signaling pathway in human B lymphocytes. CD40 is a receptor on the surface of many immune cells, including B lymphocytes. CD154, a protein, resides on the cell surface of T helper lymphocytes. This CD154-CD40 interaction results in B cell activation leading to B cell growth, survival, and antibody production. This novel finding suggests that Zn may function as an immunoregulatory nutrient to protect against infections and other chronic inflammatory diseases, such as insulin resistance and type 2 diabetes (T2D), by modulating immune function.
Objective 2 was to discover novel functions of vitamin B12 related to energy, carbohydrate (CHO) and 1-C metabolism by measuring metabolomic responses to vitamin B12 supplementation of B12 deficient humans. ARS researchers hypothesized that B12 supplementation of people with poor B12 status would alter pathways of the citric acid (TCA) cycle, mitochondrial function, FAs, 1-C, amino acid and CHO metabolism. Serum was analyzed from 27 Chilean elderly (73 years) with low serum B12 (<120 pmol/L) at baseline, before and four months after injection with 10 mg B12. B12 status was defined by serum B12, homocysteine, methylmalonic acid, holotranscobalamin and their combination (cB-12). Metabolomic response to treatment was assessed using targeted and untargeted metabolomics, and compared to the metabolome of 18 controls. Treatment improved B12 status and increased plasmalogens, phospholipids and carnitines. These and sphingomyelins were strongly correlated with B12 status. Changes in methylmalonyl CoA (MMA) and total homocysteine (tHcy) were associated with changes in acylcarnitines (TCA cycle intermediates). More interesting were strong links between cB12 and plasmalogens and other phospholipids which are major constituents of the myelin sheath of nerves and myelin in the brain. Faster peripheral conduction in the right sural nerve was correlated with plasmalogens and phosphatidyl carnitines. These associations, revealed for the first time, could explain some effects of B12 status on neurological and cognitive function. This is the first ever report of effects of B12 on the serum metabolome. This objective is completed.
Objective 3 was to measure and validate novel functional biomarkers of Zn and vitamin B12 status in response to supplementation of deficient human subjects. The Zn study is a collaboration with Children’s Hospital of Oakland Research Institute, Oakland, California. ARS researchers measured triglycerides (TG), free FAs and cholesterol in 64 blood samples from human subjects. Metabolomic analyses of these samples, including FA composition and oxylipins, are completed. All proposed analyses including statistics are completed. The results show that dietary Zn deficiency increased blood TG, but not cholesterol and free FA levels. The objective is completed. The vitamin B12 emphasis in Objective 3 was to improve and evaluate a marker of B12 status that combines the 4 single markers commonly used to evaluate B12 status; plasma B12, tHcy, methylmalonic acid and holotranscobalamin. The single biomarkers often give different estimates of B12 deficiency and are not very sensitive for detecting effects of B12 status on functions such as nerve conduction.
ARS researchers collaborated with a scientist from Aarhus University, Denmark, to revise his original model for the combined markers and produce a new indicator called “cB12”. cB12 can now be derived using two, three or four of the usual single markers, and adjusted for co-existing poor folate status. New cut-points for B12 deficiency and marginal status were established by modeling data from published studies (including those of the Davis researchers) of the relationship between cB12, hematology and cognitive outcomes in elderly. Severe B12 deficiency results in impaired nerve function and clinical signs such as neuropathy (numbness and tingling in arms and legs), but it has been difficult to show poor nerve function in less severe deficiency. ARS researchers applied their cB12 indicator in a pre- and post-treatment study in 51 Chilean elderly (age 73 years) with low serum B12 (<120 pmol/L) but no clinical symptoms. Four months post treatment, all B12 status biomarkers and conduction in the myelinated left and right sural nerves had improved and 10 new sensory action potentials appeared. Participants with high serum folate at baseline (above median of 33.9 nmol/L), a level that often occurs where flour and foods are fortified with folic acid, had less improvement in cB12.
In conclusion, the research showed that asymptomatic Chilean elderly with poor B12 status had improved conductivity in myelinated peripheral nerves after B12 treatment. The negative interaction with folate status was detected only by cB12. cB12 has now been used to diagnose B12 status in patients with gastrectomy and cancer, and assess effects of B12 supplements on cognitive performance in elderly in the United Kingdom and maternal and infant status in India. It is recommended in the National Institutes of Health (NIH) BOND (Biomarkers of Nutrition for Development) report on B12, and by the U.S. Agency for International Development (USAID). This objective is complete.
Objective 4 was to evaluate in human intervention trials the impact of dairy consumption on measures of bone, endocrine and immune function. The original research plan was to include 300 subject samples with pre- and post-study matched pairs, from a randomized controlled trial in Australia. However, once laboratory samples were analyzed, only 106 subjects had matched paired samples that were also within the limits set to show study protocol was maintained, e.g., glucose in fasting blood. Additionally, dietary intake data indicated no difference in dairy intake between the 2 groups (2 servings/day versus 4 servings/day); thus, there were no statistical effects of treatment on cardiovascular disease outcomes. Consequently, this project was closed in December 2018.
Objective 5 is to establish Reference Values for micronutrient (MN) concentrations in milk from well-nourished women who are not taking multiple MN supplements or consuming highly fortified foods. Setting Dietary Reference Values for lactating women, infants and young children requires data on the nutrient composition of human milk, but the quantity and quality of this data is very poor. With support from the Bill & Melinda Gates Foundation (BMGF), ARS researchers developed valid, efficient methods for analyzing MN in human milk, including liquid chromatography-mass spectrometry (LC-MS) to measure 5 B-vitamins and their vitamers simultaneously. They showed human milk MN are low where maternal dietary quality is poor. A Bangladesh study showed variability in milk MN was larger among mothers than was caused by time or method of milk collection, so opportunistic milk samples can represent usual MN concentrations. Supplementation increased most milk MN. In other collaborations, ARS researchers measured effects of interventions on milk MN including a B12 flour fortification program in Cameroon, thiamin fortified fish sauce in Cambodia, deworming and vitamin A in Peru, and lipid-based and multiple MN supplements in 5 countries. In November 2016, the ARS researchers received additional funding from the BMGF to develop global Reference Values for nutrients in human milk. A total of 1,000 well-nourished mother-infant dyads are being studied by collaborators in Denmark, The Gambia, Brazil and Bangladesh, and samples analyzed in Davis. Since vitamin-mineral supplements taken by most pregnant American women affect milk nutrients, the study could not be conducted in the U.S. Colostrum is collected and milk and blood three times to 8.5 months postpartum. Infants are exclusively breast-fed to 3.5 months. The Manual of Procedures is maintained in Davis. Data are entered into a REDCap database in The Gambia. Recruitment started in 2017/2018 and sample collection ends late 2020. Ongoing analyses include milk macronutrients and MN and future analyses will include oligosaccharides, milk and serum metabolomics, the infant fecal microbiome, and milk volume using deuterated water. The final product will be percentiles of milk nutrient concentrations which can be used by public health and research professionals to evaluate the quality of human milk in different locations and to assess the need for, and effectiveness of, interventions to improve maternal nutritional status.
Accomplishments
1. Zinc deficiency affecting weight and glucose metabolism is modulated by the genetic background in mice. Obesity is a serious public health problem that is highly associated with insulin resistance and impaired glucose metabolism. Genetics has been shown to play a role in obesity. Although many genetic factors in the susceptibility risk to develop obesity are identified, a genetic risk factor that links zinc deficiency to weight was completely unknown. ARS researchers in Davis, California, identified a segment of a mouse genome that influenced weight during zinc deficiency. Four candidate genes for weight regulation during zinc deficiency were discovered. This finding is novel, which may provide useful information for development of a biomarker panel for prediction and diagnosis of zinc deficiency as well as for genetic risk assessments of obesity. The research findings contribute to the health of the U.S. population as well as people living in other countries.
Review Publications
Huang, L., Tepaamorndech, S., Kirschke, C.P., Cai, Y., Zhao, J., Cao, X., Rao, A. 2019. Subcongenic analysis of a quantitative trait locus affecting body weight and glucose metabolism in zinc transporter 7 (znt7)-knockout mice. BioMed Central (BMC) Genetics. 20:19. https://doi.org/10.1186/s12863-019-0715-2.
Garrod, M.G., Buchholz, B.A., Miller, J.W., Haack, K.W., Green, R., Allen, L.H. 2018. Vitamin B12 added as a fortificant to flour retains high bioavailability when baked in bread. Nuclear Instruments and Methods in Physics Research. 438:136-140. https://doi.org/10.1016/j.nimb.2018.05.042.
