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

Abstract

O ur understanding of how Zn is metabolized in animals and humans is advancing rapidly by examination of membrane proteins that facilitate Zn transport. Zn transporters fall into two gene families, SLC30A and SLC39A (1). These families are commonly referred to as ZnT and Zip transporters, respectively.Zn metabolism has been defined partially in studies with humans and animals by using radioactive and stable isotopes (reviewed in ref. 2). Kinetic analyses and metabolic modeling have established the major pathways by which this micronutrient is processed and how organ systems produce an effective homeostatic control over absorption and excretion. The current Dietary Reference Intake (Institute of Medicine of the National Academies,Washington, DC) recommendations for Zn intake by humans are based on a balance between intestinal absorption and endogenous losses by using these metabolic assessments (3).Dietary Zn restriction leads to an up-regulation of the mediated component of the Zn-absorptive pathway in rodents, presumably by means of changes programmed in intestinal cells (4). Loss of endogenous Zn from intestinal and pancreatic secretions are concomitantly reduced during Zn restriction (reviewed in refs. 5 and 6). Urinary losses of Zn are low, indicative of high reabsorptive capacity, and they are refractory to change over a wide Zn-intake range (6). Furthermore, metabolic studies have identified organ systems that influence Zn metabolism and its response to physiologic stimuli, including hormones, cytokines, and growth factors (reviewed in ref. 5). Induction of metallothionein (MT) gene expression, which is usually concurrent with enhanced cellular Zn acquisition, has been integrated into all of these aspects of cellular Zn trafficking.Zn transporters are essential components of systems that influence Zn trafficking in times of dietary depletion or excess, such as during acute and chronic physiologic stress (e.g., infection and inflammation) and during pregnancy and lactation. Therefore, experiments with mice were designed to show the differential expression of ZnT and Zip transporter genes associated with dietary Zn restriction and excess. The data presented here identify the transporters critical for regulation of Zn homeostasis in intestinal absorptive cells, as well as pancreatic acinar cells. Materials and MethodsMice and Treatments. Young adult male CD-1 strain mice (Charles River Breeding Laboratories) were housed and fed as described in detail in refs. 7 and 8. Mice were fed one of three dietary Zn levels [Zn Ϫ (Ͻ1 mg͞kg), Zn normal (ZnN; 30 mg͞kg), or Zn ϩ (150 mg͞kg)], which represent depleted, adequate, or excessive intakes of Zn, respectively. Mice were fed individually for up to 21 days. Blood was collected by cardiac puncture under halothane anesthesia for measurement of the serum Zn concentration (7), and preparation of peripheral blood mononuclear cells (PBMCs) was performed by using NycoPrep 1.077 (Life Technologies, Grand Island, NY) gradient centrifugation. MT knockout mice and appropriate 1...

Differential impact of zinc deficiency on phagocytosis, oxidative burst, and production of pro-inflammatory cytokines by human monocytes

“…19 In zinc deficient cells, Zip-transporters responsible for the movement of zinc into the cytosol are upregulated, while transporters of the ZnT-type needed for the reverse transport are downregulated. 8,[20][21][22][23][24] In our hands zinc is removed from the culture medium by CHELEX with over 90% efficiency. The remainder might still be sufficient for the cells to maintain free zinc, despite a state of general zinc deficiency.…”

Interleukin-6 regulates the zinc transporter Zip14 in liver and contributes to the hypozincemia of the acute-phase response

“…All RNA was treated with DNase (Ambion, Austin, TX) before analysis. Sequences for transporter and other genes were obtained from GenBank, and primer͞probe sets were designed by using PRIMER EXPRESS software (Version 2.0, Applied Biosystems) and were reported previously (15,16). The primers and probe set for both Zip14 variants are presented in Table 2, which is published as supporting information on the PNAS web site.…”

“…The C-terminal cysteine was added to facilitate both crosslinking to keyhole limpet hemocyanin for antigen production in rabbits and for conjugation to SulfoLink (Pierce) for affinity purification (16). Liver was fixed with formalin and processed as described (16). Tissue sections (5 M) were incubated with the Zip14 affinity-purified primary antibody, with IgG-Alexa 594 conjugate as the secondary antibody.…”

“…It was commercially synthesized and analyzed by mass spectrometry to assess purity. The C-terminal cysteine was added to facilitate both crosslinking to keyhole limpet hemocyanin for antigen production in rabbits and for conjugation to SulfoLink (Pierce) for affinity purification (16). Liver was fixed with formalin and processed as described (16).…”

“…Fluorescence detection used TaqMan chemistry and one-step reverse transcriptase reactions (Applied Biosystems) for realtime quantitative PCR. Relative quantitation used five log 10 standard curves with 18S rRNA as the normalizer (16,17). Fluorescence was measured with a Bio-Rad iCycler detection system.…”