Location: Obesity and Metabolism Research
2024 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
This is the final report for project 2022-51000-005-000D, titled, "Molecular Determinants of Energy Metabolism Regulation and Gut Function in Response to Zinc Deficiency", which has been replaced by bridging project 2032-10700-001-000D. The following is a summary of all the objectives and sub-objectives over the life of the project. For additional information, see the new bridging project report.
In support of Sub-objective 1A, ARS researchers in Davis, California, mapped DNA sequences, also called response elements for zinc, in the promoter region of FABP3 (fatty acid bind protein 3) that bind to transcription factors to regulate the production of FABP3 for fatty acid shuttling between organelles within muscle cells and controlling fatty acid uptake into cells. Increase in FABP3 expression is associated with risks of insulin resistance, type 2 diabetes, and myocardial diseases. Moreover, ARS researchers demonstrated that a DNA fragment at about one kilobase upstream of the transcription site of FABP3 contains a zinc responsive element that regulates the expression of message RNA (mRNA) of FABP3. The researchers also show that zinc ions can turn the gene transcription on or off via DNA methylation on the zinc responsive element.
In support of Sub-objective 1B, ARS researchers in Davis, California, discovered that ZnT8, a zinc transporter mainly expressed in the pancreatic beta-cell for insulin crystallization, is also expressed in the stomach and its expression is partially overlapping with that of somatostatin (Sst), an inhibitory peptide hormone that regulates secretion of ghrelin, glucagon-like-peptide 1 (GLP1), glucose-dependent insulinotropic polypeptide (GIP), insulin, and glucagon. These hormones work together to control blood glucose homeostasis during fasting and fed conditions. The ARS researchers also demonstrated that lack of ZnT8 expression in the Sst-secretory D cells increases the density of the dense cores in the secretory granules of the D cells. Moreover, to investigate the interplay of ZnT8 and Sst in the control of glucose and insulin metabolism, the researchers established several mouse knockout (KO) lines, including ZnT8 KO, Sst KO, and a double KO of ZnT8 and Sst (DKO). These mice were fed on a regular chow diet or challenged with a high-fat diet. The results indicate that the DKO in the mouse genome negatively affect weight gains and basal insulin (fasting state) and oral glucose-stimulated insulin secretion without alterations in islet morphology or numbers compared to the wild type, ZnT8 KO or Sst KO control when they were maintained on a regular chow diet. On the other hand, the DKO mice developed severe obesity with leptin resistance, accompanied by insulin resistance and disrupted GLP and glucagon secretion after the high-fat challenge. The researchers conclude that ZnT8 may also be involved in regulating fat mass and leptin secretion besides insulin secretion, a novel discovery. A manuscript was submitted to Nutrition & Genes for publication.
In additional support of Sub-objective 1B, ARS researchers in Davis, California, discovered that lack of maternal exposure to somatostatin (Sst) induces severe insulin resistance and obesity with reduced leptin sensitivity in male offspring fed a high-fat diet. It is known that the prenatal environment impacts offspring’s risks to type 2 diabetes in adulthood. However, the effect of maternal somatostatin deficiency on offspring’ glucose and insulin metabolism have been poorly addressed. The researchers conducted a mouse study using animals, including Sst KO offspring obtained from the dams with a null-Sst genotype and Sst KO or wild type offspring obtained from the dams with a Sst heterozygous genotype. Mice were then either maintained on a regular chow diet or challenged with a high-fat diet for 15 weeks after weaning. The results show that an absence of Sst exposure in the uterus predisposes the offspring from the Sst KO dams to diet-induced obesity, insulin resistance, and leptin resistance later in adult life compared to the Sst KO and wild type offspring from the heterozygous SstKO dams. A manuscript is in preparation.
ARS researchers in Davis, California, made significant progress in support of Objective 1 on how a common genetic variation of rs13266634 (R325) in ZnT8 affects glucose-induced insulin secretion in healthy human subjects. ZnT8 is a dominant zinc transporter that provides zinc for insulin to condense to crystals stored in pancreatic beta-cells. In humans, abnormalities in ZnT8 function, such as over or under desired activity of ZnT8 due to genetic variations in the gene, can affect insulin metabolism, which is associated with risks of type 2 diabetes. The researchers prepared genomic DNA from the blood samples collected from 349 studied subjects and performed SNP (single nucleotide polymorphism) genotyping. The results suggest that the effect of this SNP on insulin and glucose metabolism in humans is gender dependent. Most importantly, the study was the first demonstrating that this SNP influences triglyceride clearance after a fat-rich diet. A manuscript was published.
ARS researchers in Davis, California, made significant progress on a subordinate project related to Objective 1, which related to this objective study topic of genetic factors on obesity, type 2 diabetes, and chronic diseases. The researchers genotyped five 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.
ARS researchers made significant progress on a microbiota project supporting Objective 2, which is 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 were published.
Also, in support of Objective 2, ARS researchers in Davis, California, conducted a feeding study to investigate how zinc affects gut microbiome of adult mice fed a mild zinc deficient diet or a control diet with an adequate zinc content containing either a low fat (10 percent kcal fat) or a high-fat (45 percent kcal fat) diet for eight weeks. During the study, mice were monitored for body weight, food intake, and fasting glucose levels. Insulin tolerance test and oral glucose tolerance test were also performed. Bacterial DNA from 144 fecal samples (collected from three time points (zero-, four-, and eight-week) after introduction of the specific diets) was extracted, PCR-amplified, and purified. Libraries were made and pooled for 16S rRNA sequencing at the U.C. Davis DNA Technologies & Expression Analysis Core. Final data is pending from the core facility.
ARS researchers also 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 made a novel discovery that defines the etiology of zinc deficiency-induced immune dysfunction leading to frequent infection or increased premature mortality in a collaborative study with French scientists. The researchers took molecular and cellular approaches identified pathogenic mutations in the human ZNT7 gene that cause zinc deficiency, bone marrow failure, stunted growth, and male hypogonadism during puberty in affected children. Most importantly, to honor the discoveries, the OMIM® - Online Mendelian Inheritance in Man® has named this genetic disease (caused by mutations in ZnT7) as Ziegler-Huang Syndrome. The results of this discovery were 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 two disease-causing mutations in a zinc transporter protein (ZNT7) that affect bone marrow health, sex maturation, and growth. A novel and significant finding of this research is that these 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. 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 scientists in Davis, California, determined the association of a common single nucleotide polymorphism (SNP) (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 blood insulin level 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 this SNP influences triglyceride clearance after a fat-rich diet that lay the groundwork for future studies in humans using this SNP as a genetic tool to identify at-risk populations of type 2 diabetes and/or obesity.
3. 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 in Davis, California, determined the association of five SNPs in APO genes with lipid markers of 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.
4. Association of estimated daily lactose consumption, lactase persistence genotype (rs4988235), and gut microbiota in healthy adults in the United States. Lactase persistence (LP) is a heritable trait in which lactose can be digested throughout adulthood. Lactase nonpersistent (LNP) individuals who consume lactose may experience microbial adaptations in response to undigested lactose. ARS scientists in Davis, California, has determined the interaction between lactose consumption, LP genotype, and gut microbiome of healthy adults in the State of California. The research indicates that consumption of dairy products by LNP individuals increases bacterial fermentation in the gut and promotes cross-feeding among bacteria capable of metabolizing lactose. The research contributes to the knowledge for understanding how nonmodifiable genotypic characteristics impact dairy product consumption. Moreover, the research suggests that it is possible for better predictive values of the lactose consumption from 24-hour recalls, which provide a valuable tool for individuals and nutritionists to estimate the impact of lactose on gut microbiome in LNP individuals and meanwhile to meet the dietary Guidelines for Americans for dairy product consumption.
5. The Znt7-null mutation has sex dependent effects on the gut microbiota and goblet cell population in the mouse colon. Zinc transporter 7 (ZnT7) is highly expressed on the membrane of the Golgi complex of intestinal epithelial cells and goblet cells (mucin secreting cells). ARS scientists in Davis, California, studied the relationship between cellular zinc homeostasis and colon mucin density as well as the gut microbiome using ZnT7 knockout mice. The research suggests that a sex-specific relationship exists among zinc homeostasis, mucin production, and microbial community compositions within the colon. ZnT7 knockout increases goblet cell numbers and mucin density in male mice, but it has little effect on gut microbiome composition. On the other hand, it is just opposite in female mice with increased proportions of the microbial taxa, Allobaculum, bacteria produce butyric acid in the colon which indirectly and positively influences the host fat storage. The findings of this study provide a fundamental knowledge of how females are more resistant to diet-induced inflammation, insulin resistance, and obesity than males in the interplay of zinc homeostasis and the gut microbiome.