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Responsive transporter genes within the murine intestinal-pancreatic axis form a basis of zinc homeostasis.

ABSTRACT: Zn homeostasis in animals is a consequence of avid uptake and retention, except during conditions of limited dietary availability, and/or factors such as parasites, which compete for this micronutrient or compromise retention by the host. Membrane proteins that facilitate Zn transport constitute the SLC30A (ZnT) and SLC39A (Zip) gene families. Because dietary recommendations are based on the balance between intestinal absorption and endogenous losses, we have studied Zn transporter expression of the murine intestinal-pancreatic axis to identify transporters that are likely to be involved in homeostatic control of Zn metabolism. Marked tissue specificity of expression was observed in Zn-depleted vs. Zn-adequate mice. As shown by quantitative PCR, Western blot analysis, and immunohistochemistry, intestinal Zip4 was markedly up-regulated in response to Zn-depletion conditions. The increased abundance of Zip4 is concentrated at the apical membrane of enterocytes. There are 16 ZnT and Zip transporters expressed in pancreas. Only two, ZnT1 and ZnT2 (both cellular Zn exporters), show a progressive down-regulation under Zn-depleted conditions. In Zn-adequate mice, ZnT1 is diffusely distributed in acinar cell cytoplasm and colocalizes with alpha-amylase but is not detected in pancreatic islets. In acinar cells during Zn depletion, ZnT1 is localized to the plasma membrane. Intestinal Zip4 up-regulation by Zn-depletion conditions is dampened in metallothionein knockout mice, suggesting that intracellular Zn pools influence these responses. The results show that Zn transporter expression in the intestinal-pancreatic axis is a component of the homeostatic regulation of this micronutrient.

SUBMITTER: Liuzzi JP

PROVIDER: S-EPMC521973 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundOverwhelming evidences suggest oxidative stress is a major cause of sperm dysfunction and male infertility. Zinc is an important non-enzymatic antioxidant with a wide range of biological functions and plays a significant role in preserving male fertility. Notably, zinc trafficking through the cellular and intracellular membrane is mediated by specific families of zinc transporters, i.e., SLC39s/ZIPs and SLC30s/ZnTs. However, their expression and function were rarely evaluated in the male germ cells. The aim of this study is to determine and characterize the crucial zinc transporter responsible for the maintenance of spermatogenesis.MethodsThe expression patterns of all 14 ZIP members were characterized in the mouse testis. qRT-PCR, immunoblot and immunohistochemistry analyses evaluated the ZIP12 gene and protein expression levels. The role of ZIP12 expression was evaluated in suppressing the sperm quality induced by exposure to an oxidative stress in a spermatogonia C18-4 cell line. Zip12 RNAi transfection was performed to determine if its downregulation altered cell viability and apoptosis in this cell line. An obese mouse model fed a high-fat-diet was employed to determine if there is a correlation between changes in the ZIP12 expression level and sperm quality.ResultsThe ZIP12 mRNA and protein expression levels were higher than those of other ZIP family members in both the mouse testis and other tissues. Importantly, the ZIP12 expression levels were very significantly higher in both mice and human spermatogonia and spermatozoa. Moreover, the testicular ZIP12 expression levels significantly decreased in obese mice, which was associated with reduced sperm zinc content, excessive sperm ROS generation, poor sperm quality and male subfertility. Similarly, exposure to an oxidative stress induced significant declines in the ZIP12 expression level in C18-4 cells. Knockdown of ZIP12 expression mediated by transfection of a ZIP12 siRNA reduced both the zinc content and viability whereas apoptotic activity increased in the C18-4 cell line.ConclusionsThe testicular zinc transporter ZIP12 expression levels especially in spermatogonia and spermatozoa are higher than in other tissues. ZIP12 may play a key role in maintaining intracellular zinc content at levels that reduce the inhibitory effects of rises in oxidative stress on spermatogonia and spermatozoa viability during spermatogenesis which help counteract declines in male fertility.

Project description:ZIP14 (slc39A14) is a zinc transporter induced in response to pro-inflammatory stimuli. ZIP14 induction accompanies the reduction in serum zinc (hypozincemia) of acute inflammation. ZIP14 can transport Zn(2+) and non-transferrin-bound Fe(2+) in vitro. Using a Zip14(-/-) mouse model we demonstrated that ZIP14 was essential for control of phosphatase PTP1B activity and phosphorylation of c-Met during liver regeneration. In the current studies, a global screening of ZIP transporter gene expression in response to LPS-induced endotoxemia was conducted. Following LPS, Zip14 was the most highly up-regulated Zip transcript in liver, but also in white adipose tissue and muscle. Using ZIP14(-/-) mice we show that ZIP14 contributes to zinc absorption from the gastrointestinal tract directly or indirectly as zinc absorption was decreased in the KOs. In contrast, Zip14(-/-) mice absorbed more iron. The Zip14 KO mice did not exhibit hypozincemia following LPS, but do have hypoferremia. Livers of Zip14-/- mice had increased transcript abundance for hepcidin, divalent metal transporter-1, ferritin and transferrin receptor-1 and greater accumulation of iron. The Zip14(-/-) phenotype included greater body fat, hypoglycemia and higher insulin levels, as well as increased liver glucose and greater phosphorylation of the insulin receptor and increased GLUT2, SREBP-1c and FASN expression. The Zip14 KO mice exhibited decreased circulating IL-6 with increased hepatic SOCS-3 following LPS, suggesting SOCS-3 inhibited insulin signaling which produced the hypoglycemia in this genotype. The results are consistent with ZIP14 ablation yielding abnormal labile zinc pools which lead to increased SOCS-3 production through G-coupled receptor activation and increased cAMP production as well as signaled by increased pSTAT3 via the IL-6 receptor, which inhibits IRS 1/2 phosphorylation. Our data show the role of ZIP14 in the hepatocyte is multi-functional since zinc and iron trafficking are altered in the Zip14(-/-) mice and their phenotype shows defects in glucose homeostasis.

Project description:Background & aimsp21-activated kinase-1 (PAK1) belongs to a family of serine-threonine kinases and contributes to cellular pathways such as nuclear factor-?B (NF-?B), mitogen-activated protein kinase (MAPK), phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT), and Wingless-related integration site(Wnt)/?-catenin, all of which are involved in intestinal homeostasis. Overexpression of PAK1 is linked to inflammatory bowel disease as well as colitis-associated cancer (CAC), and similarly was observed in interleukin (IL)10 knockout (KO) mice, a model of colitis and CAC. Here, we tested the effects of PAK1 deletion on intestinal inflammation and carcinogenesis in IL10 KO mice.MethodsIL10/PAK1 double-knockout (DKO) mice were generated and development of colitis and CAC was analyzed. Large intestines were measured and prepared for histology or RNA isolation. Swiss rolls were stained with H&E and periodic acid-Schiff. Co-immunoprecipitation and immunofluorescence were performed using intestinal organoids, SW480, and normal human colon epithelial cells 1CT.ResultsWhen compared with IL10 KO mice, DKOs showed longer colons and prolonged crypts, despite having higher inflammation and numbers of dysplasia. Crypt hyperproliferation was associated with Notch1 activation and diminished crypt differentiation, indicated by a reduction of goblet cells. Gene expression analysis indicated up-regulation of the Notch1 target hairy and enhancer of split-1 and the stem cell receptor leucin-rich repeat-containing G-protein-coupled receptor 5 in DKO mice. Interestingly, the stem cell marker olfactomedin-4 was present in colonic tissue. Increased ?-catenin messenger RNA and cytoplasmic accumulation indicated aberrant Wnt signaling. Co-localization and direct interaction of Notch1 and PAK1 was found in colon epithelial cells. Notch1 activation abrogated this effect whereas silencing of PAK1 led to Notch1 activation.ConclusionsPAK1 contributes to the regulation of crypt homeostasis under inflammatory conditions by controlling Notch1. This identifies a novel PAK1-Notch1 axis in intestinal pathophysiology of inflammatory bowel disease and CAC.

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Responsive transporter genes within the murine intestinal-pancreatic axis form a basis of zinc homeostasis.

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