Location: Obesity and Metabolism Research2020 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.
In support of Sub-objective 1A, research is focused on determining DNA binding sites for transcription factors that affect the expression of the gene Fatty Acid Binding Protein 3 (FABP3). In addition to the identification of Transcription Factor (TF) binding sites, preliminary experiments began to determine the TF binding sites that are zinc responsive in muscle cells. To accomplish these experiments, we employed a classic molecular approach in seven muscle cell lines. These cells were transfected with genomic DNA fragments that encompass various lengths of DNA sequences upstream of the transcription start site of FABP3. Positive and negative control cell lines have been developed to rigorously test our hypothesis. ARS scientists have begun to characterize these muscle cell lines. These initial experiments have demonstrated that a DNA fragment about 1,000 bases upstream of the transcription site of FABP3 contained a DNA element responsive to cellular zinc levels. These results are foundational to the continuing research for Sub-objective 1A in this project. In support of Sub-objective 1B, research continued to illustrate function of Zinc Transporter 8 (ZNT8) in regulation of somatostatin secretion from delta-cells. Somatostatin can regulate the function of a number of endocrine cells such as glucagon secretion from alpha-cells and insulin secretion in beta-cells. ARS scientists have established at least 12 insulinemia cell lines with either increased expression or reduced expression of ZNT8. Functional tests for the effect of ZNT8 on somatostatin secretion in these cell lines will be performed in the future.
1. ZNT8 allelic insufficiency provides protective effects in diet-induced glucose intolerance. ZNT8 is a dominant zinc transporter (like a vehicle in a cell) that provides zinc ions for insulin to condense to a crystal sphere in the insulin-secreting beta-cell, a type of endocrine cell in the pancreas. 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 development. Using a mouse model of human ZNT8 allelic insufficiency, ARS scientists at Davis, California, have demonstrated that individuals who carry a non-functional copy of ZNT8 could potentially be protected from diet-induced glucose intolerance. Moreover, the scientists showed that this protective effect is likely sex-dependent and involves regulation of glucagon secretion and lipid metabolism. This study lays the groundwork for a national diabetes prevention and/or treatment strategy using ZNT8 as a genetic tool to identify at-risk populations.