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ARS Home » Pacific West Area » Davis, California » Western Human Nutrition Research Center » Obesity and Metabolism Research » Research » Research Project #430814

Research Project: Molecular Determinants of Energy Metabolism Regulation and Gut Function in Response to Zinc Deficiency

Location: Obesity and Metabolism Research

2021 Annual Report

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.

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 Sub-objective 1A, ARS researchers at Davis, California, have determined the DNA length for messenger ribonucleic acid (mRNA) transcription control region of the fatty acid binding protein 3 gene and sequenced some cloned DNA pieces that contained an on/off control switch for the transcription (making mRNA) in response to changes in zinc levels within muscle cells. Preliminary DNA methylation sites in this control region were established in several DNA clones. In support of Sub-objective 1B, ARS researchers at Davis, California, have established several rodent pancreatic cell lines which potentially expressed various levels of zinc transporter 8 (ZnT8) proteins which will be used to study the function of ZNT8 in regulation of a metabolic hormone, somatostatin (Sst) which regulates glucose and insulin metabolism. Besides cellular models, in support of Sub-objective 1B, the researchers established several mouse models in which ZnT8 and/or Sst was deleted separately or together from the mouse genome. These mouse models will be used to study the interplay of zinc and hormones in regulating glucose and insulin metabolism as well as triglyceride and fatty acid levels related to type 2 diabetes and obesity. The researchers have also begun to phenotype metabolic profiles in these animal models. In support of Objective 2, ARS researchers submitted and received approval for an animal protocol by the Institutional Animal Care and Use Committee (IACUC) of University of California, Davis. This study is aiming to illustrate the effect of dietary zinc on the gut microbiome using mouse models.

1. The effect of common genetic variation of rs13266634 in ZnT8 on insulin levels depends on sex in healthy humans. ZnT8 is a dominant zinc transporter that provides zinc for insulin to condense to crystals stored in beta-cells. In humans, abnormalities in ZnT8 function, such as over-or under-desired activity of ZnT8 due to genetic variations in the gene, could affect insulin metabolism, which is associated with body glucose usage and type 2 diabetes. ARS researchers at Davis, California, have determined the association of a common Single Nucleotide Polymorphism (SNP) ID rs13266634 in ZnT8 with insulin and glucose levels before and after a high-fat meal challenge. The researchers discovered that the common allele (R325) of the SNP affected insulin during fasting in healthy subjects. Importantly, the ARS researchers found out that the effect was sex-dependent. Most importantly, the study was the first demonstrating that the SNP influences triglyceride clearance after a fat-rich diet which may lay the groundwork for future studies in humans for using this SNP as a genetic tool to identify at-risk populations for type 2 diabetes and/or obesity.

Review Publications
Kable, M.E., Riazati, N., Kirschke, C.P., Zhao, J., Tepaamorndech, S., Huang, L. 2020. The Znt7-null mutation has sex dependent effects on the gut microbiota and goblet cell population in the mouse colon. PLoS ONE. 15(9). Article e0239681.
Chin, E.L., Huang, L., Bouzid, Y.Y., Kirschke, C.P., Durbin-Johnson, B., Baldiviez, L.M., Bonnel, E.L., Keim, N.L., Korf, I., Stephensen, C.B., Lemay, D.G. 2019. Association of lactase persistence genotypes (rs4988235) and ethnicity with dairy intake in a healthy U.S. population. Nutrients. 11(8):1860.