Zinc deficiency in rats
of zinc transporters in adult rats
Pfaffl MW & Windisch W. (2003)
J Trace Elem Med Biol. 2003;17(2):97-106
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 microg Zn/g fortified with pure phytate in Zn deficiency rats and 58 microg 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.
Development of zinc deficiency in 65Zn labeled, fully grown rats
as a model for adult individuals
Windisch W. (2003)
J Trace Elem Med Biol. 2003;17(2): 91-96
Department of Livestock Sciences, BOKU - University of Natural Resources and Applied Life Sciences, Vienna, Austria. firstname.lastname@example.org
The development of zinc deficiency in adults was studied in a metabolism experiment involving 31 adult, female rats labeled homogenously with 65Zn. The animals were fed restricted amounts (8 g/day) of a semisynthetic diet containing either 58 microg Zn/g (control, n = 7) or 2 microg Zn/g (Zn deficiency, n = 24). Control animals were sacrificed at day 0 (n = 3) and day 29 (n = 4). Zinc deficient animals were sacrificed at day 1, 2, 4, 7, 11, 16, 22, and 29 (3 animals per group). The development of zinc deficiency comprised 4 phases: (I) Fecal Zn excretion needed several days to adjust to the low level of Zn intake. The high initial Zn loss via feces was counterbalanced mainly by Zn mobilization from the skeleton. (II) During the 2nd week of deficiency Zn mobilization from tissue storage changed transiently to soft tissues (mainly muscle and fat tissue). (III) After the 2nd week the skeleton resumed to mobilize Zn. (IV) At the end of the study the skeleton Zn storage was exhausted and alkaline phosphatase activity indicated severe Zn deficiency. Urinary Zn excretion was too small to contribute quantitatively to changes in Zn metabolism during any phase of Zn deficiency. In conclusion, adults may compensate a deficient Zn supply by mobilizing tissue Zn for several weeks: The skeleton revealed to be the major short-term as well as long-term source of whole body tissue Zn that can be mobilized.
Elevated caspase-3 and Fas mRNA expression in jejunum of adult rats
during subclinical zinc deﬁciency
Andrea Didiera, Wilhelm Windisch & Michael W. Pfafﬂ
Journal of Trace Elements in Medicine and Biology 18(1): 41 - 45
The programmed cell death - so-called apoptosis - is a physiological process occurring in all multicellular organisms to control cell-number homeostasis. Nevertheless, increase of apoptotic cell death in different organs can lead to pathological alterations. As zinc is a potent inhibitor of apoptosis, we investigated the inﬂuence of zinc deﬁciency on mRNA expression levels of caspase-3 and Fas in adult rats. For this purpose, 24 adult rats fed a Zn-deﬁcient diet for up to 29 days were compared to seven animals in the control group. After 1, 2, 4, 7, 11, 16, 22 and 29 days of treatment three animals were sacriﬁced (n ¼ 24). Total RNA extraction from thymus, liver, jejunum and colon was carried out. Samples were reverse transcribed and subjected to real-time PCR. Relative quantiﬁcation of caspase-3 and Fas mRNA expression was achieved on the basis of normalisation by glycerolaldehyd-3-phosphate-dehydrogenase mRNA expression levels in all samples. In jejunum, up to day 11 the relative mRNA expression of the respective genes decreased. A signiﬁcant increase in caspase-3 and Fas expression was found from day 11 of zinc deﬁciency onward. In contrast, mRNA expression in liver and colon remained unaffected, whereas thymus showed a slight but not signiﬁcant increase in the expression of these genes. This study provides the ﬁrst evidence that even moderate zinc deﬁciency in an adult, non-growing rat model is able to elevate mRNA expression levels of factors involved in early stages of apoptosis.
Effect of zinc deficiency on the mRNA
expression pattern in liver and jejunum of adult rats:
Pfaffl, B. Gerstmayer, A. Bosio, Wilhelm Windisch
Physiology, Department of Animal Sciences,Centre of Life and Food
85354 Freising, Germany
In the study presented here, the
effect of zinc deficiency on mRNA expression levels in liver and
jejunum of adult rats was analyzed. Feed intake was restricted to
8 g/day. The semi-synthetic diet was fortified with pure phytate and
contained either 2 g Zn/g (Zn deficiency, n 6) or 58 g Zn/g
(control, n 7). After 29 days of Zn depletion feeding, entire jejunum
and liver were retrieved and total RNA was extracted. Tissue specific
expression pattern were screened and quantified by microarray analysis
and verified individually via real-time RT-PCR. A relative
quantification was performed with the newly developed Relative
Expression Software Tool © on numerous candidate genes which
showed a differential expression. This study provides the first
comparative view of gene expression regulation and fully quantitative
expression analysis of 35 candidate genes in a non-growing Zn deficient
adult rat model. The expression results indicate the existence of
individual expression pattern in liver and jejunum and their tissue
specific regulation under Zn deficiency. In addition, in jejunum a
number of B-cell related genes could be demonstrated to be suppressed
at Zn deficiency. In liver, metallothionein subtype 1 and 2 (MT-1 and
MT-2) genes could be shown to be dramatically repressed and therefore
represent putative markers for Zn deficiency. Expression results imply
that some genes are expressed constitutively, whereas others are highly
regulated in tissues responsible for Zn homeostasis.
Figure 1: Representative example of a gene expression pattern captured as an image of a cDNA-array hybridised with Cy3-labelled control sample (green fluorescence) and Cy5-labelled sample (Zn deficiency in red fluorescence). Each of the 1001 cDNAs is spotted either in quadrant A and B. Four replicates for each cDNAs are spotted, resulting in four A and B quadrants, respectively. A magnification for the most up-regulated (MT-1 and MT-2) and down-regulated (IL-6R-beta) gene transcripts is shown.
Figure 2: Frequency and level of down- or up-regulation of regulated genes of microarray experiments in liver and jejunum of Zn deficiency rats. Frequency plot of both tissue expression pattern exhibit a three parametric Gaussian distribution (p <0.0001). Mean (µ) and boarders of confidential interval are indicated (µ ± 1.96 times the standard deviation of the Gaussian distribution). Significantly different expressed genes (p<0.05) were selected outside the 95% confidential interval. Lines indicate an approximation of 95% interval in liver and jejunum.
Identification of Genes Responsive to Intracellular Zinc Depletion in the HumanZinc is essential for the structural and functional integrity of cells and plays a pivotal role in the control of gene expression. To identify genes with altered mRNA expression level after zinc depletion, we employed oligonucleotide arrays with approximately 10,000 targets and used the human colon adenocarcinoma epithelial cell line HT-29 as a model. A low intracellular zinc concentration caused alterations in the steady-state mRNA levels of 309 genes at a threshold factor of 2.0. Northern blot analysis and/or real-time RT-PCR confirmed the array results for 12 of 14 selected targets. Genes identified as regulated based on microarray data encode mainly proteins involved in central pathways of intermediary metabolism (79 genes) including protein metabolism (21). We also identified five groups of genes important for basic cellular functions such as signaling (30), cell cycle control and growth (15), vesicular trafficking (15), cell-cell interaction (13), cytoskeleton (10) and transcription control (19). The latter group comprises several zinc finger-containing transcription factors of which the Kruppel-like factor 4 showed the most pronounced changes. Western blot analysis confirmed the increased expression level of this protein in cells grown under low zinc conditions. Our findings in a homogenous cell population demonstrate that the molecular mechanisms by which cellular functions are altered at a low zinc status, occur via pleiotropic effects on gene expression. In conclusion, the pattern of zinc-affected genes may represent a reference for further studies to define the zinc regulon in mammalian cells.
Colon Adenocarcinoma Cell Line HT-29.
Kindermann B, Doring F, Pfaffl M, Daniel H.
J Nutr. 2004 Jan;134(1): 57-62.
Molecular Nutrition Unit and. Department of Animal Sciences, Technical
University of Munich, D-85350 Freising-Weihenstephan, Germany.