 Zinc deficiency in rats Influence of zinc deficiency on the mRNA expression 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. wilhelm.windisch@boku.ac.at 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: Michael W.
Pfaffl, B. Gerstmayer, A. Bosio, Wilhelm Windisch
Institute of
Physiology, Department of Animal Sciences,Centre of Life and Food
Sciences, TUM,
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 Human
Zinc 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.  |
