Influence of zinc deficiency on the mRNA expression of zinc transporters in adult rats

Abstract

The accumulation of zinc in the cell is a sum of influx and efflux processes via transporter proteins, like the four Zn transporters (ZnT1–4), the divalent cation transporter 1 (DCT1) and of storage processes mainly bound to metallothionein (MT). To study the effect of Zn deficiency on mRNA expression levels, adult rats were used as an animal model. Food intake was restricted to 8 g/day containing 2 g Zn/g fortified with pure phytate in Zn deficiency rats and 58 g Zn/g in controls (n = 7). At day 1, 2, 4, 7, 11, 16, 22, and 29 of Zn deficiency, 3 animals were sacrificed, respectively (n = 24). Zn deficiency was evident from reduced plasma Zn, plasma alkaline phosphatase activity and severe mobilization of Zn from tissue stores (mainly skeleton), while food intake and body weight remained unaffected. Tissues representing Zn absorption (jejunum, colon), Zn storage and utilization (muscle, liver), and Zn excretion (kidney) were retrieved. Total RNA contents increased in colon ( p = 0.003) and trend to decrease in liver (p = 0.086). Zn deficiency was without effect on tissue total RNA concentrations in muscle tissue and kidney. Real-time reverse transcription (RT) polymerase chain reaction (PCR) assays were developed and a relative quantification on the basis of GAPDH was applied. Assays allowed a relative and accurate quantification of mRNA molecules with a sufficiently high sensitivity and repeatability. All known Zn transporter subtypes were found in the tissues. ZnT3 was newly elucidated and sequenced in rat tissues. Expression patterns and reactions to Zn deficiency were specific for the tissue analysed. Expression results imply that some transporters are expressed constitutively, whereas others are highly regulated in tissues responsible for Zn homeostasis. The most distinct changes of expression levels were shown in colon which can therefore be postulated as a highly Zn sensitive tissue. MT was down-regulated in all tissues, massively in liver ( p < 0.001) and in colon ( p = 0.002) and in tendency also in the jejunum and kidney. In parallel with intracellular Zn status it is a potent candidate gene for Zn deficiency. ZnT1 and ZnT2 showed a significant up-regulation of mRNA expression in colon ( p = 0.032 and p = 0.026) and for ZnT2 a trend of down regulation in jejunum ( p = 0.098). This study provides the first comparative view of regulation of gene expression and fully quantitative expression analysis of all known Zn transporters in a non growing adult rat model on a constant platform and therefore allows a direct comparison.