Metal transporter SLC39A14/ZIP14 modulates regulation between the gut microbiome and host metabolism.

Metal transporter SLC39A14/ZIP14 is localized on the basolateral side of the intestine, functioning to transport metals from blood to intestine epithelial cells. Deletion of Slc39a14/Zip14 causes spontaneous intestinal permeability with low-grade chronic inflammation, mild hyperinsulinemia, and greater body fat with insulin resistance in adipose. Importantly, antibiotic treatment reverses the adipocyte phenotype of Slc39a14/Zip14 knockout (KO), suggesting a potential gut microbial role in the metabolic alterations in the Slc39a14/Zip14 KO mice. Here, we investigated the hypothesis that increased intestinal permeability and subsequent metabolic alterations in the absence of Zip14 could be in part due to alterations in gut microbial composition. Dietary metals have been shown to be involved in the regulation of gut microbial diversity and composition. However, studies linking the action of intestinal metal transporters to gut microbial regulation are lacking. We showed the influence of deletion of metal transporter Slc39a14/Zip14 on gut microbiome composition and how ZIP14-linked changes to gut microbiome community composition are correlated with changes in host metabolism. Deletion of Slc39a14/Zip14 generated Zn-deficient epithelial cells and luminal content in the entire intestinal tract, a shift in gut microbial composition that partially overlapped with changes previously associated with obesity and inflammatory bowel disease (IBD), increased the fungi/bacteria ratio in the gut microbiome, altered the host metabolome, and shifted host energy metabolism toward glucose utilization. Collectively, our data suggest a potential predisease microbial susceptibility state dependent on host gene Slc39a14/Zip14 that contributes to intestinal permeability, a common trait of IBD, and metabolic disorders such as obesity and type 2 diabetes.NEW & NOTEWORTHY Metal dyshomeostasis, intestinal permeability, and gut dysbiosis are emerging signatures of chronic disorders, including inflammatory bowel diseases, type-2 diabetes, and obesity. Studies in reciprocal regulations between host intestinal metal transporters genes and gut microbiome are scarce. Our research revealed a potential predisease microbial susceptibility state dependent on the host metal transporter gene, Slc39a14/Zip14, that contributes to intestinal permeability providing new insight into understanding host metal transporter gene-microbiome interactions in developing chronic disease.

Intestine-specific deletion of metal transporter Zip14 (Slc39a14) causes brain manganese overload and locomotor defects of manganism.

Impaired manganese (Mn) homeostasis can result in excess Mn accumulation in specific brain regions and neuropathology. Maintaining Mn homeostasis and detoxification is dependent on effective Mn elimination. Specific metal transporters control Mn homeostasis. Human carriers of mutations in the metal transporter ZIP14 and whole body Zip14-knockout (WB-KO) mice display similar phenotypes, including spontaneous systemic and brain Mn overload and motor dysfunction. Initially, it was believed that Mn accumulation due to ZIP14 mutations was caused by impaired hepatobiliary Mn elimination. However, liver-specific Zip14-KO mice did not show systemic Mn accumulation or motor deficits. ZIP14 is highly expressed in the small intestine and is localized to the basolateral surface of enterocytes. Thus, we hypothesized that basolaterally localized ZIP14 in enterocytes provides another route for the elimination of Mn. Using wild-type and intestine-specific Zip14-KO (I-KO) mice, we have shown that ablation of intestinal Zip14 is sufficient to cause systemic and brain Mn accumulation. The lack of intestinal ZIP14-mediated Mn excretion was compensated for by the hepatobiliary system; however, it was not sufficient to maintain Mn homeostasis. When supplemented with extra dietary Mn, I-KO mice displayed some motor dysfunctions and brain Mn accumulation based on both MRI imaging and chemical analysis, thus demonstrating the importance of intestinal ZIP14 as a route of Mn excretion. A defect in intestinal Zip14 expresssion likely could contribute to the Parkinson-like Mn accumulation of manganism.NEW & NOTEWORTHY Mn-induced parkinsonism is recognized as rising in frequency because of both environmental factors and genetic vulnerability; yet currently, there is no cure. We provide evidence in an integrative animal model that basolaterally localized ZIP14 regulates Mn excretion and detoxification and that deletion of intestinal ZIP14 leads to systemic and brain Mn accumulation, providing robust evidence for the indispensable role of intestinal ZIP14 in Mn excretion.

Hepatic ZIP14-mediated zinc transport is required for adaptation to endoplasmic reticulum stress.

Extensive endoplasmic reticulum (ER) stress damages the liver, causing apoptosis and steatosis despite the activation of the unfolded protein response (UPR). Restriction of zinc from cells can induce ER stress, indicating that zinc is essential to maintain normal ER function. However, a role for zinc during hepatic ER stress is largely unknown despite important roles in metabolic disorders, including obesity and nonalcoholic liver disease. We have explored a role for the metal transporter ZIP14 during pharmacologically and high-fat diet-induced ER stress using Zip14-/- (KO) mice, which exhibit impaired hepatic zinc uptake. Here, we report that ZIP14-mediated hepatic zinc uptake is critical for adaptation to ER stress, preventing sustained apoptosis and steatosis. Impaired hepatic zinc uptake in Zip14 KO mice during ER stress coincides with greater expression of proapoptotic proteins. ER stress-induced Zip14 KO mice show greater levels of hepatic steatosis due to higher expression of genes involved in de novo fatty acid synthesis, which are suppressed in ER stress-induced WT mice. During ER stress, the UPR-activated transcription factors ATF4 and ATF6α transcriptionally up-regulate Zip14 expression. We propose ZIP14 mediates zinc transport into hepatocytes to inhibit protein-tyrosine phosphatase 1B (PTP1B) activity, which acts to suppress apoptosis and steatosis associated with hepatic ER stress. Zip14 KO mice showed greater hepatic PTP1B activity during ER stress. These results show the importance of zinc trafficking and functional ZIP14 transporter activity for adaptation to ER stress associated with chronic metabolic disorders.

The Multiple Faces of the Metal Transporter ZIP14 (SLC39A14).

The SLC39A family of metal transporters was identified through homologies with the Zrt- and Irt-like (ZIP) proteins from yeast and plants. Of all the ZIP transporters, ZIP14 is arguably the most robustly characterized in terms of function at the integrative level. Mice with a global knockout of Zip14 are viable, thus providing the opportunity to conduct physiologic experiments. In mice, Zip14 expression is highly tissue specific, with the greatest abundance in the jejunum > liver > heart > kidney > white adipose tissue > skeletal muscle > spleen > pancreas. A unique feature of Zip14 is its upregulation by proinflammatory conditions, particularly increased interleukin 6 (IL-6) and nitric oxide. The transcription factors AP-1, ATF4, and ATF6α are involved in Zip14 regulation. ZIP14 does not appear to be zinc-regulated. The Zip14 knockout phenotype shows multiple sites of ZIP14 function, including the liver, adipose tissue, brain, pancreas, and bone. A prominent feature of the Zip14 ablation is a reduction in intestinal barrier function and onset of metabolic endotoxemia. Many aspects of the phenotype are accentuated with age and accompany increased circulating IL-6. Studies with 65Zn, 59Fe [nontransferrin-bound iron (NTBI)] and 54Mn show that ZIP14 transports these metals. At a steady state, the plasma concentrations of zinc, NTBI, and manganese are such that zinc ions are the major substrate available for ZIP14 at the cell surface. Upregulation of ZIP14 accounts for the hypozincemia and hepatic zinc accumulation associated with acute inflammation and sepsis and is required for liver regeneration and resistance to endoplasmic reticulum (ER) stress. Zip14 ablation in mice produces a defect in manganese excretion that leads to excess manganese accumulation in the brain that produces characteristics of Parkinsonism.

Dietary Zinc Regulates Apoptosis through the Phosphorylated Eukaryotic Initiation Factor 2α/Activating Transcription Factor-4/C/EBP-Homologous Protein Pathway during Pharmacologically Induced Endoplasmic Reticulum Stress in Livers of Mice.

BACKGROUND: Several in vitro studies have shown that zinc deficiency could induce endoplasmic reticulum (ER) stress, resulting in activation of the unfolded protein response. OBJECTIVE: We aimed to determine whether consumption of a zinc-deficient diet (ZnD) triggers ER stress and to understand the impact of dietary zinc intake on ER stress-induced apoptosis using a mouse model. METHODS: Young adult (8-16 wk of age) male mice of strain C57BL/6 were fed either a ZnD (<1 mg/kg diet), or a zinc-adequate diet (ZnA; 30 mg/kg diet). After 2 wk, liver, pancreas, and serum samples were collected and analyzed for indexes of ER stress. In another experiment, mice were fed either a ZnD, a ZnA, or a zinc-supplementation diet (ZnS; 180 mg/kg diet). After 2 wk, vehicle or tunicamycin (TM; 2 mg/kg body weight) was administered to mice to model ER stress. Liver and serum were analyzed for indexes of ER stress to evaluate the effects of zinc status. RESULTS: Mice fed a ZnD did not activate the apoptotic and ER stress markers in the liver or pancreas. During the TM challenge, mice fed a ZnD showed greater C/EBP-homologous protein expression in the liver (3.8-fold, P < 0.01) than did ZnA-fed mice. TM-treated mice fed a ZnD also had greater terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick-end labeling-positive cells in the liver (2.2-fold, P < 0.05), greater hepatic triglyceride accumulation (1.5-fold, P < 0.05), greater serum alanine aminotransferase activity (1.6-fold, P < 0.05), and greater protein-tyrosine phosphatase 1B activity (1.5-fold, P < 0.05), respectively, than did those fed a ZnA. No significant differences were observed in these parameters between mice fed ZnAs and ZnSs. CONCLUSIONS: Consumption of a ZnD per se is not a critical factor for induction of ER stress in mice; however, once ER stress is triggered, adequate dietary zinc intake is required for suppressing apoptotic cell death and further insults in the liver of mice.

Influence of ZIP14 (slc39A14) on intestinal zinc processing and barrier function.

ZIP14 is a zinc transport protein with high expression in the small intestine and liver. Zip14 is upregulated during endotoxemia and leads to increased liver zinc content and transient hypozinemia. Since body zinc status and inflammation are associated with changes in intestinal permeability, we hypothesized that ZIP14 may influence intestinal permeability. Wild-type (WT) and Zip14 knockout (KO) mice were used to determine ZIP14-associated intestinal zinc metabolism and effects on permeability. Fractionation of plasma membranes revealed that ZIP14 is localized to the basolateral membrane of enterocytes. Studies utilizing (65)Zn administered by subcutaneous injection revealed greater zinc accumulation in the SI of Zip14 KO mice compared with WT mice. Isolation of endosomes confirmed the presence of ZIP14. Quantification of endosomal zinc concentration by FluoZin-3AM fluorescence demonstrated that zinc is trapped in endosomes of Zip14 KO mice. Intestinal permeability assessed both by plasma FITC-dextran following gavage and by serum endotoxin content was greater in Zip14 KO mice. Threonine phosphorylation of the tight junction protein occludin, which is necessary for tight junction assembly, was reduced in KO mice. Claudin 1 and 2, known to have an inverse relationship in regards to tight junction integrity, reflected impaired barrier function in KO jejunum. These data suggest involvement of ZIP14 in providing zinc for a regulatory role needed for maintenance of the intestinal barrier. In conclusion, ZIP14 is a basolaterally localized protein in enterocytes and is involved in endosomal trafficking of zinc and is necessary for proper maintenance of intestinal tight junctions.

Tolerance to graded dosages of histidine supplementation in healthy human adults.

BACKGROUND: Histidine is an essential amino acid with health benefits that may warrant histidine supplementation; however, the clinical safety of histidine intake above the average dietary intake (1.52-5.20 g/d) needs to be vetted. OBJECTIVES: We aimed to determine the tolerance to graded dosages of histidine in a healthy adult population. METHODS: Healthy adults aged 21-50 y completed graded dosages of histidine supplement (4, 8, and 12 g/d, Study 1) (n = 20 men and n = 20 women) and/or a 16-g/d dosage of histidine (Study 2, n = 21 men and n = 19 women); 27 participants (n = 12 men and n = 15 women) completed both studies. After study enrollment and baseline measures, participants consumed encapsulated histidine for 4 wk followed by a 3-wk recovery period. Primary outcomes included vitals, select biochemical analytes, anthropometry, serum zinc, and body composition (via DXA). RESULTS: No changes in vitals or body composition occurred with histidine supplementation in either study. Plasma histidine (measured in subjects who completed all dosages for Studies 1 and 2) was elevated at the 12- and 16-g/d dosages (compared with 0-8 g/d, P < 0.05) and blood urea nitrogen increased with dosage (P = 0.013) and time (P < 0.001) in Study 1 and with time in Study 2 (P < 0.001). In Study 1, mean ferritin concentrations were lower in 12 g/d (46.0 ng/mL; 95% CI: 34.8, 60.9 ng/mL) than in 4 g/d (51.6 ng/mL; 95% CI: 39.0, 68.4 ng/mL; P = 0.038). In Study 2, 16 g/d increased mean aspartate aminotransferase from baseline (19 U/L; 95% CI: 17, 22 U/L) to week 4 (24 U/L; 95% CI: 21, 27 U/L; P < 0.001) and mean serum zinc decreased from baseline (0.75 µg/dL; 95% CI: 0.71, 0.80 µg/dL) to week 4 (0.70 µg/dL; 95% CI: 0.66, 0.74 µg/dL; P = 0.011). CONCLUSIONS: Although values remained within the normal reference ranges for all analytes measured, in all dosages tested, the human no-observed adverse effect level was determined to be 8 g/d owing to changes in blood parameters at the 12-g/d dosage.This trial was registered at clinicaltrials.gov as NCT04142294.

Proteomic analysis shows the upregulation of erythrocyte dematin in zinc-restricted human subjects.

BACKGROUND: Although the importance of adequate zinc intake has been known for decades, the estimated global prevalence of zinc deficiency remains high. This substantiates the need for a specific and sensitive status assessment tool. OBJECTIVE: The objective was to evaluate erythrocyte zinc transporters as candidate molecules with the potential of being a biomarker of dietary zinc status in humans. DESIGN: A 24-d observational study with acclimation (7 d, 10.4 mg Zn/d), zinc-depletion (10 d, 0.3 mg Zn/d), and zinc-repletion (7 d, 29.5 mg Zn/d) phases was conducted in healthy men (n = 9). Proteomic approaches including Western blot analyses and tandem mass spectrometry were implemented to identify the zinc responsiveness of selected red blood cell membrane proteins. RESULTS: Zinc transporter 1 (ZnT1) and Zrt/Irt-like proteins ZIP8 and ZIP10 were detected in human erythrocyte membranes. No effects of short-term dietary zinc depletion were observed on the amounts of these proteins. However, changes in a cytoskeletal protein, dematin, by zinc depletion were identified through the nonspecific signals produced by an anti-ZIP8 antibody. This response was further validated by a dematin-specific antibody and with erythrocytes collected from mice fed a zinc-deficient diet. CONCLUSIONS: The presence of ZnT1, ZIP8, and ZIP10 in human red blood cells implicates their role in the regulation of cellular zinc metabolism in the human erythroid system. The zinc responsiveness of membrane dematin suggests its capability to serve as a biomarker for dietary zinc depletion and its involvement in impaired erythroid membrane fragility by zinc restriction. This trial was registered at clinicaltrials.gov as NCT01221129.

Plenary Lecture 2: Transcription factors, regulatory elements and nutrient-gene communication.

Dramatic advances have been made in the understanding of the differing molecular mechanisms used by nutrients to regulate genes that are essential for their biological roles to carry out normal metabolism. Classical studies have focused on nutrients as ligands to activate specific transcription factors. New interest has focused on histone acetylation as a process for either global or limited gene activation and is the first mechanism to be discussed. Nuclear ATP-citrate lyase generates acetyl-CoA, which has been shown to have a role in the activation of specific genes via selective histone acetylation. Transcription factor acetylation may provide a second mode of control of nutrient-responsive gene transcription. The third mechanism relates to the availability of response elements within chromatin, which as well as the location of the elements in the gene may allow or prevent transcription. A fourth mechanism involves intracellular transport of Zn ions, which can orchestrate localized enzyme inhibition-activation. This process in turn influences signalling molecules that regulate gene expression. The examples provided in the present review point to a new level of complexity in understanding nutrient-gene communication.

Zinc transporter ZIP8 (SLC39A8) and zinc influence IFN-gamma expression in activated human T cells.

The zinc transporter ZIP8 is highly expressed in T cells derived from human subjects. T cell ZIP8 expression was markedly up-regulated upon in vitro activation. T cells collected from human subjects who had received oral zinc supplementation (15 mg/day) had higher expression of the activation marker IFN-gamma upon in vitro activation, indicating a potentiating effect of zinc on T cell activation. Similarly, in vitro zinc treatment of T cells along with activation resulted in increased IFN-gamma expression with a maximum effect at 3.1 microM. Knockdown of ZIP8 in T cells by siRNA decreased ZIP8 levels in nonactivated and activated cells and concomitantly reduced secretion of IFN-gamma and perforin, both signatures of activation. Overexpression of ZIP8 by transient transfection caused T cells to exhibit enhanced activation. Confocal microscopy established that ZIP8 is localized to the lysosome where ZIP8 abundance is increased upon activation. Loss of lysosomal labile zinc in response to activation was measured by flow cytometry using a zinc fluorophore. Zinc between 0.8 and 3.1 microM reduced CN phosphatase activity. CN was also inhibited by the CN inhibitor FK506 and ZIP8 overexpression. The results suggest that zinc at low concentrations, through inhibition of CN, sustains phosphorylation of the transcription factor CREB, yielding greater IFN-gamma expression in T cells. ZIP8, through control of zinc transport from the lysosome, may provide a secondary level of IFN-gamma regulation in T cells.