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Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City, KS 66160-7421
3To whom correspondence should be addressed. E-mail: gandrews{at}kumc.edu.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • mice • metallothionein • pregnancy • transgenic • pancreas • zinc homeostasis
Understanding the mechanisms by which zinc homeostasis is achieved is an area of active investigation. A deficiency of this essential trace element in humans and laboratory rodents impairs many fundamental biological processes (1
–3
) including embryonic and fetal development (4
–8
), the ability to fight disease (8
–11
), carcinogenesis (12
), and taste, vision and other brain functions (13
–16
). The diverse effects of zinc deficiency reflect the fact that this metal is important for the proper structure and function of a large number of proteins.
All organisms have adapted mechanisms to regulate the uptake, efflux and storage of zinc, and several mammalian genes involved in these processes have been identified (17
–19
). Integral membrane proteins, members of a zinc transporter superfamily (ZnT1–6),4 which efflux zinc from cells or function in the vesicular storage of zinc inside of cells have been reported (19
–21
). In addition, members of the ZIP family of membrane-spanning zinc transporters are also found in mammals and appear to be responsible for zinc uptake (18
). The human genetic disease of zinc metabolism, acrodermatitis enteropathica (22
,23
), was recently mapped to the human ZIP4 gene (24
–26
). Thus, the expression and regulation of several zinc transport proteins is central to zinc homeostasis in higher eukaryotes.
The cysteine-rich metallothioneins (MT) also play a role in mammalian zinc homeostasis (27
). MT are abundant intracellular zinc-binding proteins that are isolated from normal mouse tissues as zinc7-complexes (28
). Furthermore, MT-I gene expression is regulated by the zinc-dependent transcription factor MTF-1 (29
). Cells exposed to zinc accumulate relatively large amounts of MT. Reciprocally, MT expression is diminished and MT protein is degraded during periods of zinc deficiency. Thus, it has been hypothesized that MT provides a biologically important labile pool of zinc in mice. This concept is supported by studies of transgenic mice that overexpress MT-I (30
) or that have loss-of-function mutations (gene knockout) in the MT-I/II genes (31
,32
). Transgenic overexpression of MT-I in mice, under the control of its own promoter, causes increased accumulation of zinc in many maternal organs, and results in significantly increased resistance to the teratogenic effects of dietary zinc deficiency during pregnancy (33
). In contrast, studies of MT-I/II knockout mice demonstrated increased sensitivity to dietary zinc deficiency during pregnancy (34
), as well as during the neonatal period (35
). The ability to maintain zinc homeostasis during pregnancy was impaired in these mice (36
), as was intestinal secretion of zinc (37
,38
) and the ability of the pancreas to retain zinc (39
).
These studies demonstrate that MT is important for proper zinc homeostasis in mice and imply possible tissue-specific roles of MT in this process. In mice, the MT genes are expressed in a cell-type specific manner in maternal and embryonic tissues (40
,41
). Among the adult organs, there are particularly high concentrations of MT in the pancreas (42
,43
), and MT has been detected in pancreatic exocrine secretions (43
). The pancreas has been implicated in zinc homeostasis (44
), and a large amount of zinc is released from the pancreas into the intestinal tract (45
). The objective of these studies was to address the roles of pancreatic MT in zinc homeostasis during pregnancy. Specifically, we sought to determine whether genetic manipulation of pancreatic MT-I levels in mice could alter the detrimental effects of maternal dietary zinc deficiency during pregnancy on embryonic development.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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All experiments involving animals were conducted in accordance with NIH guidelines for the care and use of experimental animals, and all animal experiments were approved by our Institutional Animal Care and Use Committee. Transgenic mice were created as described below, using eggs obtained from C57Bl/6 females bred with SJL males. Transgenic mice were bred to CD-1 females (48–60 d old; Charles River Breeding Laboratories, Raleigh, NC) for three generations. The heterozygous transgenic mice obtained were then inbred to yield homozygous transgenic mice and nontransgenic control littermates. Those mice were then inbred to create a working colony of animals, and age-matched mice of each strain were compared.
Mouse diets were all purchased from Harlan Teklad (Madison, WI) and have been described in detail previously (33
). Zinc levels in the diets were as follows: zinc-deficient (Zn-D), 7.7–15.4 µmol/L (1 µg/g); zinc-adequate (Zn-A), 769 µmol/L (50 µg/g). These diets each contained 
18 µg/g Cu, and were otherwise identical.
Experimental designs.
Experiments were initially designed to examine the organ specificity and extent of overexpression of MT-I in transgenic mice relative to control mice. In addition, the effects of pancreatic overexpression of MT-I on zinc levels in the pancreas were determined. Mice in these experiments were fed the Zn-A diet. At least six female mice per group (age 40–60 d) were killed and each pancreas, kidney, liver and intestine was harvested, rapidly frozen in liquid nitrogen and later assayed in sextuplet for Cd-binding activity (MT) and zinc content. Similarly, liver, intestine and kidney were harvested from at least 6 mice per group, and samples were pooled and frozen in liquid nitrogen for subsequent RNA extraction and Northern blot analysis. Also, pancreas samples from these mice were processed immediately for RNA extraction and Northern blotting rather than being frozen.
Experiments were then designed to determine whether pancreatic overexpression of MT-I in these transgenic mice attenuates the detrimental effects of maternal dietary zinc deficiency on embryonic development. These experiments were conducted according to Experimental Design 4 published previously (34
). Homozygous transgenic female mice and control female mice (40–60 d old) were mated with CD-1 male mice. On d 1 (vaginal plug) of pregnancy, mice were placed in pairs in cages with stainless steel false bottoms to reduce recycling of zinc (46
). Mice had free access to the Zn-A feed and deionized-distilled water. Water bottles were washed in 4 mol/L HCl and rinsed in deionized water to remove zinc (46
). On d 8, the diet was changed to the Zn-D diet (or where indicated, mice continued to consume the Zn-A diet), and effects on embryonic development were examined on d 14. The total number of implantation sites and the number of resorbed and nonresorbed implantation sites per uterus were determined visually, and the morphology of nonresorbed fetuses was determined by examination under a dissecting microscope.
Further experiments were designed to determine whether pancreatic overexpression of MT-I in these transgenic mice attenuates other sensitive measures of zinc deficiency, such as the loss of maternal pancreatic MT and alterations in the expression of zinc-regulated genes in the embryonic visceral yolk sac. Tissues were collected on d 14 of pregnancy from transgenic mice (Zn-A or Zn-D; n = 6), treated as described above. Individual maternal pancreata were frozen in liquid nitrogen and later assayed in sextuplet for Cd-binding activity. Embryonic visceral yolk sacs were collected and pooled from implantation sites, frozen in liquid nitrogen and later assayed by Northern blot hybridization for relative levels of MT-I and ZIP4 mRNAs, as measures of embryonic zinc status (33
,47
).
Creation of elastase-MT-I transgenic mice.
The rat elastase I promoter and the human growth hormone 3'-untranslated region (UTR) and polyadenylation signal were provided by Dr. Ray MacDonald (University of Texas Health Sciences Center, Dallas, TX). The proximal 500-bp of this promoter is sufficient to direct cell-specific gene expression in pancreatic acinar cells in transgenic mice (48
–50
). The mouse MT-I gene was provided by Dr. Richard Palmiter (University of Washington, Seattle, WA). This gene is 1 kb in length and the protein coding region is divided into three exons (51
). A rat elastase promoter/MT-I gene/human growth hormone 3'-UTR fusion gene was engineered. In this construct, the rat elastase promoter (-500 to + 8 bp) was ligated to the mouse MT-I gene at +17. The 617-bp human growth hormone 3'-UTR and polyadenylation signal (+1540 to +2157) (50
) was ligated to the MT-I gene at +953 bp, just after the translation stop codon (Fig. 1
). This 2-kb fusion gene was released from the plasmid vector and purified from an agarose gel using a Qiagen Gel Extraction Kit (Valencia, CA) and further purified by phenol/chloroform/isoamyl alcohol extractions. DNA was ethanol-precipitated and redissolved in microinjection buffer (10 mmol/L Tris-HCl, 0.25 mmol/L EDTA, pH 7.4). DNA concentration was measured by fluorometry and diluted to 1 ng/µL for microinjection. DNA microinjection and manipulation were conducted at the Institutional Transgenic and Gene Targeting Facility at the University of Kansas Medical Center.
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PCR was used to determine the genotype of DNA from mice using reaction conditions described previously (52
). Genotyping of elastase-MT-I transgenic mice was performed using antisense-strand primer (GAGTCTTACCGGTGGAGCAGGAG) beginning at +88 bp in the mouse MT-I gene (53
) and a sense-strand primer (CTTGGGTTAACTGAGTGCCGGCC) beginning at -219 bp in the rat elastase I promoter (48
). Homozygosity of transgenic mice was confirmed by outbreeding to CD-1 mice and PCR genotyping of the offspring.
Northern blot detection of MT-I and ZIP4 mRNAs.
Tissue RNAs were isolated as described in detail previously (47
,54
). Total RNA (3 µg) was size fractionated by agarose-formaldehyde gel electrophoresis, transferred and cross-linked to nylon membranes (55
). Northern blots membranes were hybridized and washed under stringent conditions as described (33
,54
,55
). Hybrids were detected by autoradiography with intensifying screens at -70°C, and quantitated by radioimage analysis (Molecular Dynamics, Sunnyvale, CA). Although not shown, duplicate gels were stained with acridine orange or the same membrane was rehybridized with a glyceraldehyde phosphate dehydrogenase (GAPDH) probe to monitor RNA loading and integrity (54
).
Mouse MT-I probe was as described (33
). The mouse GAPDH probe was cloned by reverse transcription (RT)-PCR (see below) of the protein coding region (Genbank accession number M32599) using mouse liver RNA. The mouse ZIP4 cDNA was identified by homology with human ZIP4 (25
) in the GenBank mouse nonredundant data base on the NCBI server as a 2264-bp cDNA (accession number AK005535). The protein coding region of ZIP4 mRNA was amplified by RT-PCR from mouse intestinal RNA using the Improm-II Reverse Transcriptase kit (Promega, Madison, WI) and Pfu polymerase (Stratagene, La Jolla, CA) for PCR. The sense primer began at +86 and the antisense primer began at + 2156 in this cDNA. Each primer was 26 bp in length. The RT-PCR product was cloned and the DNA sequence confirmed. Probes were labeled using the Random Primers DNA Labeling System according to the manufacturer’s instructions (Invitrogen, Carlsbad, CA). Probes had specific activities ranging from 10 to 30 GBq/µg.
Cadmium-hemoglobin exchange assay for MT (Cd-binding proteins).
Steady-state levels of total MT were quantitated using the cadmium-hemoglobin exchange assay (56
).
Atomic adsorption spectrophotometry.
Samples were weighed, vacuum dried and then digested in nitric acid. Zinc was quantitated in the acid extract using a Perkin Elmer 540 flame atomic adsorption spectrophotometer. Bovine liver (Standard Reference Material 1577b; National Institute of Standards and Technology; Gaithersburg, MD) was used as a reference standard for zinc content.
Statistical analysis.
Data were analyzed using ANOVA and Student’s t test (Student-Newman-Keuls method). Differences were considered significant when P < 0.05. Values are reported as means ± SEM.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Transgenic mice in which the mouse MT-I gene is expressed under the control of the rat elastase I promoter were created (Fig. 1)
. The rat elastase I promoter has been shown to direct pancreatic acinar cell-specific expression of the human growth hormone gene in transgenic mice. In initial experiments, 11 transgenic founder lines were created using the intact mouse MT-I gene downstream of the elastase promoter. Although several of these transgenic lines displayed low level expression of the transgene in the pancreas, none had detectably elevated MT levels (data not shown). As shown previously (33
), MT levels in the normal mouse pancreas were greater than those in the kidney, intestine and liver (Table 1
).
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and one line displayed increased MT concentration in the pancreas (Table 1)
. This transgenic mouse strain was bred to homozygosity and examined in detail. The transgenic mice had elevated MT levels in the pancreas, but not in the liver, intestine or kidney compared with the control, nontransgenic mice. MT levels in the pancreas of the transgenic mice varied among individuals, but were five to seven times higher than those in the control pancreas (also see Fig. 2
). Northern blot hybridization suggested that MT-I mRNA in the pancreas was also more abundant in the transgenic than in the control mice (Fig. 2)
, but was not more abundant in the other organs examined. In this experiment, MT-I mRNA levels in the liver of the transgenic mice also appeared elevated relative to those in the liver of the nontransgenic control mice. However, this was not reproducible and MT protein levels were not elevated in the liver of these transgenic mice. Among the organs examined, MT mRNA was most abundant in the pancreas of the transgenic mice. No further attempt was made to determine the relative abundance of the endogenous MT-I mRNA transcripts compared with the transgene transcripts in these mice.
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Pancreatic overexpression of MT-I correlates with decreased sensitivity to dietary zinc deficiency during pregnancy.
In either transgenic or control mice fed the Zn-A diet during pregnancy, essentially no abnormal embryos were detected on d 14 (data shown for control mice only). In contrast, the zinc deficiency created in this experiment resulted in impaired growth, delayed and/or abnormal development and separation of the digits in the d-14 mouse embryo. In the control mice, 21.3% of the embryos exhibited morphological defects (Table 2
). In contrast, only 5.8% of the embryos in the elastase-MT-I transgenic females had developed abnormally by d 14.
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MT levels in the pancreas of the transgenic mice declined >95% during zinc deficiency (Fig. 3A
). MT levels were highly variable but were detectable in the transgenic zinc-deficient mice. MT levels in the pancreas from control mice also declined >95% under these conditions (data not shown), as reported previously (33
).
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). As shown in Figure 3
B, MT-I mRNA levels were depressed and ZIP4 mRNA levels were elevated in the visceral yolk sacs taken from transgenic mice during maternal dietary zinc deficiency. |
DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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) or not to express MT-I/II genes (31
,32
) suggest that one of the multitude of functions of these proteins is in zinc metabolism (27
). Involvement in the secretion, retention and tissue distribution of zinc in various organs has been reported. For example, MT may reduce the efficiency of zinc uptake by the intestine (38
,57
), but increase pancreatic and hepatic retention of this essential metal (39
,58
). In the absence of MT, more zinc is released into the gut. Thus, although the MT-I/II genes are nonessential, they serve to protect mice against periods of zinc excess and deficiency (33
–36
,59
).
Although the consequences of global alterations in MT-I/II gene expression in mice have been examined in some detail, as discussed above, the effects of tissue-specific alterations in the expression of these genes have not. It has been reported that overexpression of MT-III, a predominantly neuronal member of the MT family of genes, inhibits the growth of cultured cells (60
), and ectopic expression in the pancreas of transgenic mice causes necrosis of that organ (61
). This is not the case for MT-I, as reported herein. Ectopic expression of MT-I in the pancreas leads to increased zinc and MT levels with no detrimental effects on that organ. This is not unexpected because the growth inhibitory activity of MT-III does not reflect its metal-binding activity but rather its unique structure (62
,63
), and transgenic mice that overexpress the MT-I gene accumulate very high levels of MT-I in the pancreas (33
) with no detrimental effects.
The pancreas is important for zinc homeostasis (44
,64
–66
), and a significant amount of zinc is released from the pancreas into the intestinal tract (45
). In mice, the MT-I/II genes are expressed in a cell-type specific manner in several maternal and embryonic tissues (40
,41
), and particularly high concentrations of MT are found in the pancreas (42
,43
) and its exocrine secretions (43
). In control mice, MT-bound zinc represents 20% of the total tissue zinc pool in the pancreas (33
). In addition, pancreatic MT gene expression and protein levels are remarkably responsive to dietary zinc (33
), and the loss of MT from the pancreas is pronounced during the organogenic period if maternal dietary zinc is limiting (33
). These findings suggest that pancreatic MT may play a role in zinc homeostasis. Results reported herein provide direct evidence in support of that concept. Overexpression of MT-I specifically in the pancreas provided measurable protection for the embryo against the effects of moderate dietary zinc deficiency during pregnancy. However, the extent of protection against zinc deficiency was relatively modest compared with that provided by the transgenic overexpression of MT-I throughout the animal (33
). In the latter transgenic mice, embryos were essentially completely protected against the effects of severe dietary zinc deficiency by midgestation. Thus, as is expected, pancreatic MT-I represents only one component of the zinc homeostatic mechanism in mice.
How pancreatic MT-I functions in zinc homeostasis is suggested by the findings that it provides a mechanism to maintain zinc in the pancreas (39
), and that its overexpression leads to increased zinc levels in this organ (33
). This pancreatic zinc-MT provides a labile pool of zinc. During periods of zinc deficiency, the pancreatic zinc-MT pool is degraded and/or excreted, which contributes to zinc efflux either into the blood stream or into the intestinal lumen. Evidence in support of the latter concept is provided by the detection of MT protein in pancreatic secretions (43
), and the observation that nearly as much zinc is lost in bile-pancreatic secretions as is absorbed by the intestine under normal dietary conditions (44
,67
). During periods of zinc deficiency, the intestinal absorption of zinc is increased (68
–70
). Thus, although the amount of pancreatic zinc-MT is small relative to that in the entire animal, it is uniquely positioned to provide a pool of zinc that can be directly secreted into and then reabsorbed by the intestines during periods of zinc deficiency (71
).
During the course of these studies, we found that MT levels in the pancreas of these transgenic mice were severely depleted by dietary zinc deficiency. This was unexpected because expression of the MT-I transgene was under control of the rat elastase-I promoter rather than the metal-regulated MT-I promoter. Thus, our studies suggest that post-transcriptional mechanisms can play an important role in regulating MT protein levels. Whether this mechanism involves destabilization of MT-I mRNA and/or protein was not examined. The stability of MT mRNA does increase during exposure of cells to zinc or cadmium (72
), and that mechanism is not understood. However, protein stability also plays a role in regulating MT levels. The turnover of MT has been shown to increase in vivo in cadmium-treated rats and cultured cells (73
), and apo-MT is more susceptible to protease cleavage than is the metallated protein (74
). During zinc deficiency, MT may be demetallated and actively degraded. Overall, these studies suggest that MT protein levels are ultimately regulated by several mechanisms, transcriptional and post-transcriptional, which may reflect a fundamental role of this protein in zinc homeostasis.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Present address: Department of Genetics, University of North Carolina, Chapel Hill, NC 27599-7264
4 Abbreviations used: GAPDH, glyceraldehyde phosphate dehydrogenase; MT, metallothionein; RT-PCR, reverse transcription-polymerase chain reaction; UTR, untranslated region; Zn-A, zinc-adequate diet; Zn-D, zinc-deficient diet; ZnT, zinc transporter; ZIP, zinc/iron regulated transporter-related zinc transporter.
Manuscript received 15 August 2002. Initial review completed 13 September 2002. Revision accepted 30 September 2002.
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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