Location: Obesity and Metabolism Research2019 Annual Report
1a. Objectives (from AD-416):
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.
1b. Approach (from AD-416):
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.
3. Progress Report:
This report documents progress for new project 2032-51000-005-00D, which continues progress from the expired research project 2023-51000-004-00D "Novel Functions and Biomarkers for Vitamins and Minerals." Objective 1A is to 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. ARS researchers in Davis, California, modified a firefly luciferase reporter vector to carry an antibiotics gene so that cell lines can be generated after introduction of the modified vector into mammalian cells. Eight genomic DNA fragments that encompass various lengths of DNA sequences upstream of the transcription start site of the Fabp3 gene have been successfully cloned into this modified plasmid vector and transfected into muscle cells. Next, the ARS researchers will use the luciferase report gene to identify critical DNA sequences and its bound proteins in the Fabp3 promoter region that controls the transcription. Objective 1B is to illustrate that cellular zinc homeostasis can indirectly control whole body glucose utilization by influencing somatostatin secretion in gut endocrine cells. The researchers have completed mRNA and protein expression analysis of insulin, somatostatin, and zinc transporter 8 (Znt8) in mouse RIN-14B cells (a mouse insulinoma cell line). Immunohistochemistry has also been completed for double-staining of Znt8 and endocrine cells in the mouse gastrointestinal tract. Work on Objective 2 will start in 2021.
Lemay, D.G., Huang, S., Huang, L., Alkan, Z., Kirschke, C.P., Burnett, D.J., Wang, Y., Hwang, D.H. 2019. Temporal changes in postprandial blood transcriptomes reveal subject-specific pattern of expression of innate immunity genes after a high-fat meal. Journal of Nutritional Biochemistry. 72:108209. https://doi.org/10.1016/j.jnutbio.2019.06.007.