Role of Zinc on Metallothionein Gene Expression in Pigs

Zinc is critical for the functional and structural integrity of cells and contributes to a number of important processes including gene expression (Cousins, 1996; O’Halloran, 1993; Silva and Williams, 1991). Pools used to supply zinc for these functions are regulated by transporters at the plasma membrane as well as at intracellular sites (McMahon and Cousins, 1998). The liver is also a key component of this metabolic response to infection and oxidative stress. Expression of metallothionein (MT), a cysteine-rich zinc-binding protein, appears to be linked to these metabolic changes (Andrews, 2000 & Davis and Cousins, 2000).  Experiments with human subjects have shown that MT expression is altered when the dietary zinc supply is restricted or supplemented. Erythrocyte MT protein concentrations, as measured by enzyme-linked immunosorbent assay (ELISA), are reduced or elevated, after a lag period of; 6 days, when the dietary zinc intake of these subjects is correspondingly adjusted (Grider et al., 1990 and Sullivan et al., 1998). Similar changes have been observed in red blood cells from zinc-deficient rats (Robertson et al., 1989)

 

Imbra and  Karin (1987) suggested that metallothionein (MT), low-molecular-weight, highly inducible, heavy-metal-binding proteins serve in the regulation of intracellular Zn metabolism. Among the Zn-requiring systems are several enzymes involved in DNA replication and repair. Therefore, during periods of active DNA synthesis there is likely to be an increased demand for Zn, which could be met by elevated MT synthesis.

 

Kimball et al. (1995) reported that zinc deficiency resulted in a lower rate of hepatic protein synthesis. The decreased rate of protein synthesis was due to a decrease in the rate of synthesis of proteins retained in the liver, with no apparent change in the synthesis of secreted proteins. Analysis of expression of specific gene products, as assessed by in vitro translation of total RNA followed by two-dimensional gel analysis, showed that the expression of only a few mRNAs was altered by zinc deficiency.

 

RNA synthesis is an essential component of gene expression and Zn2+ ions are required for catalytic activity of the RNA polymerases (RNA nucleotide transferases which are Zn metallo-enzymes) (Cousins, 1998 a).

 

Cousins (1998 b) reported that experimental animals show suspended growth when their diet is severely restricted in Zn content .The resulting lack of growth could be caused by a lack of Zn required for gene expression. However, the cessation of growth is more likely to be a secondary response to a stimulus to restrict the energy-requiring processes needed for additional cell replication and protein synthesis.

 

Zinc regulates the gene expression machinery. It affects the structure of chromatin, the template function of its DNA, the activity of numerous transcription factors and of RNA polymerases. Hence, it determines both the types of mRNA transcripts synthesized and the rate of transcription itself (Falchuk1998).

 

Martinez et al. (2004) investigated the effect of dietary Zn and phytase on relative MT mRNA abundance and protein concentration in newly weaned pigs. They fed diets containing adequate (150 mg Zn/kg) or pharmacological concentrations of Zn (1000 or 2000 mg Zn/kg), as zinc oxide, with or without phytase (0, 500 phytase units (FTU)/kg, Natuphos, BASF) were fed in a 3×2 factorial design.  They reported that hepatic and renal relative MT mRNA abundance and protein were greater (P<0.05) in pigs fed 1000 mg Zn/kg with phytase, or 2000 mg Zn/kg with or without phytase vs. the remaining treatments. Intestinal mucosa MT mRNA abundance and protein were greater (P<0.05) in pigs fed 2000 mg Zn/kg with phytase than in pigs fed 2000 mg Zn/kg alone or 1000 mg Zn/kg with phytase. They conclude that feeding 1000 mg Zn/kg with phytase enhances MT mRNA abundance and protein and Zn absorption to the same degree as 2000 mg Zn/kg with and without phytase.

 

Chang et al. (2005) reported that zinc deficiency in rats enhances esophageal cell proliferation, causes alteration in gene expression, and promotes esophageal carcinogenesis. Zinc replenishment rapidly induces apoptosis in the esophageal epithelium thereby reversing cell proliferation and carcinogenesis. To identify zinc-responsive genes responsible for these divergent effects, they did oligonucleotide array-based gene expression profiling analyses in the precancerous zinc-deficient esophagus and in zinc replenished esophagi after treatment with intragastric zinc compared with zinc-sufficient esophagi. Thirty-three genes (21 up-regulated and 12 down-regulated) showed a 2-fold change in expression in the hyperplastic zinc-deficient versus zinc-sufficient esophageal epithelia. Expression of genes involved in cell division, survival, adhesion, and tumorigenesis were markedly changed. The zinc-sensitive gene metallothionein- 1 (MT-1 was up-regulated 7-fold, the opposite of results for small intestine and liver under zinc-deficient conditions. Keratin 14 (KRT14, a biomarker in esophageal tumorigenesis), carbonic anhydrase II (CAII, a regulator of acid-base homeostasis), and cyclin B were up-regulated >4-fold. Immunohistochemistry showed that metallothionein and keratin 14 proteins were overexpressed in zinc-deficient esophagus, as well as in lingual and esophageal squamous cell carcinoma from carcinogen-treated rats, emphasizing their roles in carcinogenesis.

 

Study was conducted to investigate the mechanism for the effect of elevated levels of dietary zinc oxide in enhancing the intestinal growth of weanling piglets by Xilong et al (2006). One group was fed the basal diet containing 100 mg Zn/kg diet. The other group was fed the basal diet supplemented with zinc oxide to provide 3000 mg Zn/kg diet. Small-intestinal mucosa collected for analyzing IGF-I and IGF-I receptor gene expression. They reported that the mRNA and protein levels for IGF-I and IGF-I receptor in the small intestine were markedly enhanced (P < 0.05) by feeding elevated levels of Zn.

 

Carlson et al. (2007) allocated pigs to four dietary treatments, consisting of low or high dietary zinc (100 or 2500 ppm) in combination with low or high dietary copper (20 or 175 ppm) in pigs. They considered lymphocyte metallothionein (MT) mRNA and intestinal mucosa MT mRNA concentrations were included as zinc status markers. They observed that the dietary zinc treatments increased the zinc concentration in plasma as well as the zinc and MT mRNA concentration in mucosa. Lmphocyte MT mRNA concentrations did not reflect the differences in dietary zinc supplementation.

 

Liver RNA from pigs fed 150, 1,000, or 2,000 mg Zn/kg, or 1,000 mg Zn/kg with phytase (n = 4 per treatment) was reverse transcribed and examined using the differential display reverse transcription polymerase chain reaction technique. Liver RNA from pigs fed 150 or 2,000 mg Zn/kg (n = 4 per treatment) was also evaluated using a 70-mer oligonucleotide microarray by Martinez et al. (2008). They concluded  that feeding pharmacological Zn (1,000 or 2,000 mg Zn/kg) affects genes involved in reducing oxidative stress and in amino acid metabolism, which are essential for cell detoxification and proper cell function.