Location: Obesity and Metabolism Research
2023 Annual Report
Objectives
The goal of the proposed objectives is to understand the underlying molecular mechanisms that link zinc to Type 2 Diabetes (T2D) and explore ways to prevent and/or reduce progression of T2D through improvement of zinc nutritional status.
Objective 1: Investigate how zinc status affects lipid and glucose metabolism.
Subobjective 1A: Determine DNA binding sites (cis-regulatory elements) for transcription factors that control Fatty acid binding protein 3 (Fabp3) transcription in response to changes in cellular zinc status.
Subobjective 1B: Cellular zinc homeostasis can indirectly control whole body glucose utilization by influencing somatostatin secretion in gut endocrine cells.
Objective 2: Determine whether the microbiome is altered by interaction with zinc status.
Approach
We hypothesize that zinc is an important regulator and gatekeeper in fatty acid uptake via regulation of Fabp3 expression in muscle cells. Thus, we seek to uncover cis-regulatory elements and their bound transcriptional factors (TFs) or methylation hot spots that are responsive to changes in cellular zinc status in or near the promoter of Fabp3. Plasmids with various lengths of the upstream of the transcription site of Fabp3 will be generated and the potential functional transcriptional factor (TF) binding sites will be revealed by luciferase reporter activity in muscle cells, including wild type (wt) and Znt7-KO muscle cells. Next, we will examine the methylation status of the promoter sequence adjacent to the transcriptional start site of Fabp3 using genomic DNA purified from muscle tissues from wt and Znt7-KO mice. DNA methylation status will be determined using EZ DNA Methylation-gold kits from Zymo Research. Additionally, we hypothesize that increase in cytoplasmic zinc levels will alter other hormone productions other than insulin. Therefore, we will perform experiments in vitro (cell lines) and in vivo (mice) to illustrate the effect of Znt8 overexpression or knockdown on hormone secretion in endocrine cells from the pancreas or the gut. RT-PCR, Western blot analysis, immunohistochemistry, and ELISA will be used in the study. Lastly, we hypothesize that consuming a zinc-rich diet will increase zinc content in the colon, which may help to develop beneficial microbial communities and promote a healthy mucosa, resulting in better resistance to diet-induced insulin resistance. B6 mice (26 mice per dietary group) will be randomized to either a low-fat diet or a Western-style diet (WD) with indicated zinc amounts in foods ranging from mild zinc deficiency to zinc supplement (the total zinc intake will vary from 67% to 200% of DRI values for rodents). The control diet contains 10% fat, 20% protein, 70% carbohydrates, 5% fiber, and the indicated amounts of zinc while the WD contains 45% fat, 20% protein, 35% carbohydrates, 5% fiber and the indicated amounts of zinc. The primary endpoints will be the changes of body zinc status, fasting blood glucose and insulin levels, Hb A1c, fasting serum triglycerides, and fasting free fatty acids as well as oral glucose tolerance and intraperitoneal insulin tolerance. The colon tissue will be collected for examining mucus layer thickness and mucin intensity, gut barrier function by measurement of endocannabinoid system tone and mRNA expression of genes involved in pro-inflammatory cytokine genes. Plasma will be isolated for zinc and inflammatory marker measurements. Fecal samples in the cecum will also be isolated for zinc determinations and the gut microbiota analysis.
Progress Report
In support of Objective 1, ARS researchers at Davis, California, established mouse models of single gene knockout of somatostatin (Sst) and Znt8 (Zinc transporter 8) knockouts (KO) as well as a double knockout of SstKO/Znt8KO. Somatostatin is an important inhibitory hormone for controlling insulin and other glucose metabolism-related hormone secretion. Znt8 is a zinc transporter involved in insulin crystallization and glucose-induced insulin secretion. The researchers finished proposed experiments, including mouse breeding, dietary challenge, and phenotyping data collection from both single gene knockout mice and double knockout mice fed either a chow diet or a high-fat diet for 15 weeks. The phenotyping data collections included weekly body weight measurements, blood glucose monitoring, insulin and glucose tolerance tests, obesity or diabetes-related blood hormone measurements, histological and immunochemical examination of the islets of the pancreas, epithelial cells of the stomach and small intestine, and the hypothalamic arcuate nucleus (where hunger and satiety are regulated via Sst and other hormones) of the brain. The researchers are drafting manuscripts.
In support of Objective 2, ARS researchers at Davis, California, completed a feeding study for mice challenged with a high-fat (45% kcal from fat) diet for 2 months. Meanwhile, the researchers also completed a parallel control feeding study for mice fed a low-fat diet (10% kcal from fat). The researchers finished the proposed experiments, including dietary challenge and phenotyping data collection. The completed phenotyping data collections included body weight and food intake measurements, as well as blood glucose measurements at time points of 0, 1, and 2 months after the indicated diet was given. The researchers also completed insulin and glucose tolerance tests in the experimental mice. Fresh fecal pellets were collected at the time points mentioned above. Fresh fecal matter in the cecum and tissues including blood, cecum and colon were collected at necropsy. Moreover, the researchers completed microbial genomic DNA purification from collected fecal samples.
The researchers made significant progress on a human phenotyping study supporting Objective 1, which related to the impact of dysregulation of cellular zinc homeostasis caused by a genetic variant in the human ZNT8 gene in the insulin secreting beta-cells of the pancreas on risk of type 2 diabetes and dyslipidemia in humans. ZNT8 is a zinc transporter that provides zinc for insulin to form dense crystals in beta-cells for storage and secretion after a meal. Over-expression of ZNT8 in diabetic beta-cells is implicated in beta-cell death that accelerates the disease progress. The researchers completed purifying genomic DNA and genotyping the variant of ZNT8 in 349 study subjects challenged with a high-fat diet. A negative association of the major C-variant of ZNT8 (major C-variant: approximately 70% of populations carry at least one copy of this variant) with blood glucose, insulin, and lipid profile was discovered, and the results were published.
Researchers made significant progress on a human phenotyping study supporting Objective 1, which related to the study of genetic factors on obesity, type 2 diabetes and chronic diseases. Researchers genotyped five single nucleotide polymorphisms (SNPs) in apolipoprotein genes (APOs), including APOA5, APOB, APOC3, and APOE and a lipoprotein receptor gene (LDLR), in 349 human subjects challenged with a high-fat liquid diet. The assessment of the impact of these SNP variants on lipid metabolism before and after the high-fat meal challenge was completed. The results demonstrated that the studied genetic variants were strongly associated with fasting and postprandial cholesterol levels, which are risk factors for cardiovascular diseases, including stroke and heart attack. The results of the findings were published.
The ARS researchers made significant progress on a subordinate project (58-2032-0-004F, IFR log 66116) supporting Objective 1, which related to the study of zinc deficiency, hormone secretion, lipid metabolism, and chronic diseases. Globally, zinc deficiency impact on human health is significant, as zinc deficiency is estimated to cause more than 450,000 deaths annually in children under five years of age. However, the etiology of illness and death is not well understood. The researchers discovered the etiology of zinc deficiency-induced immune dysfunction leading to frequent infection or increased premature mortality due bone marrow failure in a collaborative study with French scientists. The researchers also discovered the underlying molecules and signaling pathways leading to cell programed death (apoptosis) in zinc deficient cells and stunted growth and male hypogonadism during puberty. The results of this discovery have been published.
ARS researchers made significant progress on a microbiota project supporting Objective 2, which related to the topic of the interaction of the gut microbiome with zinc homeostasis. The researchers discovered that zinc deficiency induced by Znt7 knockout in mice significantly reduced goblet cell numbers leading to decreased mucus layer thickness in the colon. The health of the gut microbiota is highly and positively associated with the mucus layer thickness of the colon, which results in improved glucose metabolism. The researchers discovered that the gut microbial community composition was linearly correlated with the mucus layer thickness of the colon in Znt7 knockout mice independent of sex. Thus, dysbiosis observed in Znt7 knockout mice is primarily associated with reduced production of mucins in the colon. The results of this discovery have been published.
Accomplishments
1. Discovery of genetic disease-causing mutations in the human ZNT7 gene. Dietary zinc deficiency causes stunted growth, impaired immune function, and frequent infection or even death. Globally, zinc deficiency impact on human health is significant as zinc deficiency is estimated to cause more than 450,000 deaths annually in children under 5 years old. However, the etiology of illness or genetic factors are not well understood. ARS researchers in Davis, California, discovered rare mutations in a zinc transporter protein (ZNT7) required for zinc homeostasis in the human body. A novel and significant finding of this research is that mutations in ZNT7 disproportionately affect blood cell productions in the bone marrow causing pancytopenia (severe lower-than-normal numbers of white and red blood cells and platelets) and secretion of growth hormone and testosterone causing stunted growth of fetus, infants, and toddlers and lack of puberty in affected males. Importantly, without early diagnosis and treatment, affected individuals can die from the disease. Similarly, severe dietary zinc deficiency can cause death in children if not treated. Thus, the ARS scientists have shed light on the fundamental mechanisms underlining the clinical presentations of zinc deficiency. Moreover, the research findings may provide a gateway for discovering novel biology with broad impact for children’s health. Furthermore, this research may even one day enable the development of sensitive and specific biomarkers for zinc deficiency and development of screening tools or drugs to treat children with defects in zinc metabolism.
2. SNPs in apolipoproteins contribute to sex-dependent differences in blood lipids before and after a high-fat dietary challenge in healthy U.S. adults. Single nucleotide polymorphisms (SNPs) in apolipoprotein (APO) genes link to dyslipidemia and type 2 diabetes. However, limited studies have determined the impact of SNPs in apolipoproteins on lipid markers and lipid clearance after a high-lipid meal challenge in healthy U.S. adults. ARS scientists at Davis, California, determined the association of 5 SNPs in APO genes with lipid markers in subjects enrolled in a cross-sectional Nutritional Phenotyping Study. The ARS scientists demonstrated that the genetic effect in the apolipoprotein genes on cholesterol metabolism is dependent on sex and body mass index in healthy subjects. These findings contribute to scientific evidence and applications for precision nutrition indicating that blood cholesterol levels are dependent on genetics and the gender in adults whereas effects of SNPs in APO genes on triglyceride metabolism is limited.
Review Publications
Yang, Z., Wang, Y.E., Kirschke-Schneide, C.P., Stephensen, C.B., Newman, J.W., Keim, N.L., Cai, Y., Huang, L. 2023. Effects of a genetic variant rs13266634 in the zinc transporter 8 gene (SLC30A8) on insulin and lipid levels before and after a high-fat mixed macronutrient tolerance test in U.S. adults. Journal of Trace Elements in Medicine and Biology. 77. Article 127142. https://doi.org/10.1016/j.jtemb.2023.127142.
Huang, L., Yang, Z., Kirschke-Schneide, C.P., Prouteau, C., Copin, M., Bonneau, D., Pellier, I., Coutant, R., Miot, C., Ziegler, A. 2023. Identification of novel compound heterozygous variants in the SLC30A7 (ZNT7) gene in two French brothers with stunted growth, testicular hypoplasia and bone marrow failure. Human Molecular Genetics. 32(12):2016-2031. https://doi.org/10.1093/hmg/ddad033.
Wang, Y.E., Kirschke-Schneide, C.P., Woodhouse, L.R., Bonnel, E.L., Stephensen, C.B., Bennett, B.J., Newman, J.W., Keim, N.L., Huang, L. 2022. SNPs in apolipoproteins contribute to sex-dependent differences in blood lipids before and after a high-fat dietary challenge in healthy U.S. adults. Biomed Central (BMC) Nutrition. 8. Article 95. https://doi.org/10.1186/s40795-022-00592-x.
James, K.L., Gertz, E.R., Kirschke-Schneide, C.P., Allayee, H., Huang, L., Kable, M.E., Newman, J.W., Stephensen, C.B., Bennett, B.J. 2023. Trimethylamine N-Oxide response to a mixed macronutrient tolerance test in a cohort of United States adults. International Journal of Molecular Sciences. 24(3). Article 2074. https://doi.org/10.3390/ijms24032074.
