The response of zinc transporter gene expression of selected tissues in a pig model of subclinical zinc deficiency

 Summary

• This study compared the relative mRNA expression of all mammal zinc (Zn) transporter genes in selected tissues of weaned piglets challenged with short-term subclinical Zn deficiency (SZD).
• The dietary model involved restrictive feeding (450 g/animalday−1) of a high-phytate diet (9 g/kg) supplemented with varying amounts of zinc from ZnSO47H2O ranging from deficient to sufficient supply levels (total diet Zn: 28.1, 33.6, 38.8, 42.7, 47.2, 67.0 mg Zn/kg).

Zn has a strong toxic potential if the concentration within a biological system exceeds a certain threshold

• Therefore, the regulation of Zn uptake, redistribution, and excretion within an organism must be tightly controlled
• A complex molecular network that modulates the expression of specific Zn transport peptides to benefit metabolic function under changing dietary and physiological conditions maintains mammalian Zn homeostasis
• 24 Zn transporters have been described in mammals, belonging to the solute carrier (SLC) families 30 (ZnT) and 39 (ZIP).
• The ZnT and ZIP transporter genes differ regarding their transport mechanism, as well as the direction of transport
• Differences in response patterns have been reported depending on the biological model used for the investigations
• It is suggested that an intestinal-pancreatic axis is responsible for the difference in the abundance of ZIP4 and ZIP1 genes
• ZIP4 is presented on the apical side of enterocytes, especially within the jejunum, whereas ZIP1 is on the basolateral side

Material and methods

• This animal study was evaluated and approved by the animal welfare officer of the faculty TUM School of Life Sciences Weihenstephan, Technical University of Munich, and further approved and registered by the responsible animal welfare authorities (District Government of Upper Bavaria, Federal State of Bavaria: case number 55.2.54-2532.63-11).
• The study design as well as communication of material, methods and results comply to the ARRIVE Guidelines.

Animals and diets

• To ensure full body Zn stores at day one of the experimental period, a basal diet (based on corn and soybean meal, dietary phytate (InsP6) concentration 9 g/kg) was supplemented with 60 mg Zn/kg from analytical-grade ZnSO4 * 7H2O to adjust the total dietary Zn at a sufficient level of 88.0 mg/kg diet.
• The basal diet was designed to meet all the recommendations of the National Research Council regarding the feeding of weaned piglets except for zinc
• Blocking parameters comprised live weight, litter mates and sex, thereby yielding a good standardization of body development and genetic background between treatment groups

Sampling conditions

• All animals were killed by exsanguination under anesthesia (azaperone and ketamine) without fasting and tissue samples were taken from jejunal and colonic mucosa as well as liver and kidney.
• Tissue samples for gene expression analyses were immediately incubated in RNAlater (Life Technologies GmbH) overnight and subsequently stored at −80°C according to the manufacturer´s instructions.

Analyses of dry matter and total zinc concentration in diets

• Dry matter was sampled in triplicate and milled through a 0.5 mm screen.
• Zn concentrations were measured by atomic absorption spectrometry (AAS) using a certified AAS Zn standard material.

Gene expression analysis

• Primer design, assay quality control, and chemical procedures were performed as described earlier
• All oligonucleotides bind to homologous regions of respective transcripts to amplify the pool of potential transcript variants within one reaction
• Reference genes were selected by applying the RefFinder tool
• The 2−∆∆Ct method was used to normalize the gene expression data
• Assays amplify sequences which appear to be highly conserved between mammal species

Statistical Analysis

• Data analysis was performed with SAS 9.5.4 (SAS Institute Inc.) applying the procedure NLMIXED to estimate linear broken-line regression models (y = a + bx + cx) based on independent group means relative to dietary Zn concentration (n = 8).
• The goodness-of-fit of linear versus polynomial models is statistically compared using F-statistics.
• Only significant regression models were applied for data presentation and interpretation in the present manuscript. A threshold of P≤.05 was considered to indicate statistical significance.

Results

• All animals remained in good health throughout the whole trial.
• The experimental model introduced finely-graded adaptions in Zn status parameters as well as the Zn concentrations in the tissues under study (Supplementary Figure 1). These data were presented earlier [36] and are therefore not described in more detail in this manuscript.

Tissue specificity

• Most of the analysed ZnT and ZIP transcripts were abundant within the tissues examined in the present study.
• Some transcripts (ZnT5, ZIP9, ZIP1, ZIP2, ZIP3, ZIP4, ZIP6, ZIP7, ZIP10, and ZIP13) were expressed in such a highly stable manner over treatment groups that they served as reference genes for data normalization.

Effects of varying dietary zinc supply on the relative ZnT and ZIP transcript abundance in examined porcine tissues of weaned piglets

• Many transcripts recognized within the jejunum, colon, liver and kidney of growing piglets showed significant dietary thresholds in response to changes in dietary Zn supply.
• The only exceptions were ZIP2 and ZIP3 in colonic tissue as well as the candidate genes that served as reference genes for data normalisation within respective tissues
• Broken-line regression models were estimated based on independent arithmetic group means relative to dietary zinc concentration.
• Parameter estimates are presented as means ± SEs to indicate the precision of estimation. P≤.05 was considered to be significant.

Discussion

• We investigated the expression of ZnT (SLC30) and ZIP transporter genes in weaned piglets exposed to different Zn concentrations in the diet (28.1 to 88.0 mg Zn/kg).

Specificity of ZnT and ZIP family member gene expression for selected porcine tissues

• Most transcripts were present in all examined tissues, which seems to agree with the available literature (summarized in [3], [4], [5], [6]). The only exceptions were ZIP10 and ZIP12, which were not recognized at all.
• ZIP10 mRNA was only expressed in nephric tissue.

Response of ZnT and ZIP family member gene expression to dietary zinc

• Previously been shown that some Zn transporter genes respond to deficient dietary Zn supply.
• Genes showing a significant dose-response to dietary ZN could be divided into two groups: those with significant breakpoints at either ~40 or ~60 mg Zn/kg diet and those with no significant breakpoint at all
• Clinical Zn deficiency is the endpoint in physiological adaption to body Zn depletion
• Zn homeostasis: organism increases Zn absorption capacity at the gut barrier during periods of malnutrition or Zn malnutrition
• ZIP4 is identified as the main active ZIP4 transporter at the apical membrane of enterocytes
• Regarding genes with a statistical breakpoint <40 mg/kg, we postulate that they may be involved in regulation of tissue Zn in connection with redox and immune functions

ZIP4 and ZnT1 gene expression in the intestine and kidney under SZD conditions

• Previous studies suggest that ZIP4 transcription directly responds to the status of whole-body Zn homeostasis through KLF4 activity
• Colonic ZIP4 expression has already been discussed
• Interestingly, we did not find any significant response of jejunal ZIP4 gene expression
• The jejunum is generally regarded as the main site of Zn absorption [69]
• We therefore expected a significant upregulation
• This was not the case, which again highlights clear differences between subclinical and clinical Zn deficiency
• Most previous studies conducted experiments for ≥2 weeks and found no significant reaction of kidney or urinary Zn to varying Zn feeding

Reference

10.1016/j.jnutbio.2020.108576