Location: Obesity and Metabolism Research
2022 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 Sub-objective 1B, researchers have established mouse models of somatostatin (Sst) and Znt8 (mouse Zinc transporter 8) knockouts (KO). 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 secretion. The researchers have completed the phenotyping experiments for Sst KO as well as Znt8 KO mice. Tissues have been collected for enzyme-linked immunosorbent assay (ELISA), immunohistochemistry, and gene and protein expression studies. Blood hormone levels, including insulin, insulin C-peptide glucagon, glucagon-like peptide 1 (GLP1), peptide YY (PYY), and leptin, have been determined for male wild type (wt) and Sst KO mice. Ribonucleic acid (RNA)-sequencing analysis for stomach tissues isolated from male wt and Sst KO mice was also completed. Mouse breeding and subsequent phenotyping experiments (mouse chow diet or high-fat diet) for double knockout Sst and Znt8 KO are ongoing.
Researchers made significant progress on a subordinate project related to Objective 1 on the impact of zinc homeostasis on obesity and type 2 diabetes. Researchers analyzed changes of cellular zinc homeostasis caused by genetic variants of a zinc transporter, ZNT8 (human Zinc transporter 8) on lipid metabolism before and after a high-fat meal challenge in human subjects. ZNT8 provides zinc for insulin to form dense crystals in the pancreas for secretion after a meal. Three hundred forty-nine study subjects were genotyped for a single nucleotide polymorphism (SNP) variant in the human ZNT8 gene. A manuscript was submitted that described the association of the SNP with fasting and postprandial clinical lipid and glucose metabolism markers, including triglycerides, total cholesterol, low-density lipoprotein-cholesterol (LDL), high-lipoprotein-cholesterol (HDL), non-esterified free fatty acid (NEFAs), glucose, insulin, HbA1c (hemoglobin A1C), and Ghrelin.
In further support of Objective 1, researchers made significant progress on a subordinate project related to genetic factors on obesity, type 2 diabetes and chronic diseases. The researchers genotyped 5 SNPs in apolipoprotein genes (APOs), including APOA5, APOB, APOC3, and APOE and a lipoprotein receptor gene (LDLR), in 349 human subjects. The impact of these SNP variants on lipid metabolism before and after a high-fat meal challenge was assessed. A manuscript describing the association of these SNPs with fasting and postprandial clinical lipid metabolism markers, including triglycerides, total cholesterol, LDL, HDL, and NEFAs was submitted to a peer-reviewed journal.
Additionally, researchers made significant progress on a subordinate project in support of Sub-objective 1B, relating to zinc deficiency, hormone secretion, lipid metabolism, and chronic diseases. Researchers functionally characterized a compound mutation in the human ZNT7 gene that cause severe phenotypes of zinc deficiency and growth retardation and a lean phenotype in humans in a collaborative study with French scientists. A manuscript was drafted and will be submitted soon.
In support of Objective 2, researchers at Davis, California, submitted and received an approval of an animal protocol by the Institutional Animal Care and Use Committee of University of California, Davis for conducting a study aiming to illustrate the effect of dietary zinc on the gut microbiome using mice fed a high- (45% kcal from fat) or a low-fat (10% kcal from fat) for two months.
Accomplishments
