M311 Physiology, School Of Biomedical and Chemical Sciences, The University of Western Australia, Crawley, Western Australia, Australia
Submitted 29 April 2005 ; accepted in final form 30 July 2005
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ABSTRACT |
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anemia; transcription; nutrition; pancreatic duodenal homeobox
It is unlikely that iron is directly required for disaccharidase activity (16) or that iron deficiency alters the kinetic properties of sucrase (5). In addition, it has been shown that many other functions of the intestinal mucosa are adversely affected by iron deficiency, including a significant decrease in the secretory component (4, 5), a decrease in the specific activity of diamine oxidase (4, 5), and an apparent inability to manufacture membrane-bound high-molecular-weight glycoproteins (20). As such, most hypotheses explaining the mechanisms of the relationship between iron status and disaccharidase activity focus on an impaired ability to manufacture all brush border glycoproteins, either at the level of initial synthesis, posttranslational modification, or trafficking to the brush border membrane (4, 5, 9, 20, 38).
However, many other important characteristics of the small intestine do not change during iron deficiency, suggesting that enterocyte function is not impaired to such a large degree. Most importantly, cell proliferation requires iron (21), yet intestinal morphology is rarely different from normal, even under severe iron deficiency (4, 9, 16, 30). More specifically, the DNA synthesis rate of intestinal enterocytes is not affected by iron deficiency (4, 27), nor is the proportion of mitotic cells in the mucosa (9). Finally, whereas the activities of some enzymes in the mucosa are reduced, the activities of folate conjugase (16) and lysosomal -galactosidase (4) remain unchanged.
On the basis of the available data, we hypothesized that the decrease in disaccharidase activity seen during iron deficiency is not caused by a reduced ability to synthesize membrane-bound glycoproteins per se but rather due to an alteration of the expression of the genes encoding the enzymes. Because sucrase and lactase mRNA expression and the presence of enzymatically active protein are considered to be markers for differentiated enterocytes (10, 36), it is possible that the iron status alters the normal pattern of differentiation as the cells migrate to the villus tip or that a specific regulatory mechanism sensitive to the effects of iron deficiency controls the abundance of disaccharidase mRNA in each individual differentiated enterocyte.
To test our hypothesis, iron-deficient and control conditions were established by dietary means in Wistar rats. The enzymatic activities of two disaccharidases, sucrase-isomaltase (sucrase) and lactase phlorizin hydrolase (lactase), were tested for both conditions as well as the activity of intestinal alkaline phosphatase (IAP)-II, because it is an inducible brush border membrane glycoprotein unrelated to carbohydrate digestion (23, 41). The kinetic properties of both sucrase and lactase were also examined to confirm that any decreases result from lower quantities of enzyme present rather than due to reduced enzyme efficiencies. The location of sucrase protein and gene expression along the crypt to villus axis was observed by immunofluorescence and in situ hybridization, respectively, so as to determine any changes in the normal pattern of enterocyte differentiation. Semiquantitative real-time PCR was performed to determine sucrase and lactase mRNA levels and assess whether decreases in enzyme quantity were due to an inability to manufacture membrane-bound glycoproteins or attributable to mechanisms that specifically control levels of disaccharidase gene expression. Finally, mRNA levels for activators of sucrase and lactase gene expression caudal-related homeobox (CDX)-2, GATA-binding protein (GATA)-4, and hepatocyte nuclear factor (HNF)-1 (3, 26, 37) and the repressor pancreatic duodenal homeobox (PDX)-1 (15, 39) were measured to gain insight into possible mechanisms by which any alterations in gene expression may be regulated by iron deficiency.
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METHODS |
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Assessment of conditions. All rats were fasted overnight before being tested but still allowed free access to distilled water. Blood was collected from animals anesthetized with 60 mg/kg pentobarbital sodium (Nembutal) by drawing directly from the left ventricle. Samples were tested for hemoglobin content [measured by a freshly calibrated Hemocue Hemoglobin meter (Helsinborg, Sweden)] and hematocrit. Iron deficiency was considered sufficient for this study if hemoglobin and hematocrit values were <65% of those measured for the control group.
Mucosa collection. Preliminary studies indicate that sucrase and lactase activities and expression in iron-deficient and control animals are highest in the proximal jejunum and that, in all cases, samples taken from this region closely correlate to whole intestine activity and expression (data not shown). As such, enzyme activities were obtained from the whole length of the intestine, whereas histological samples and gene expression data were obtained from the proximal jejunum.
The entire length of the small intestine was carefully removed from the pylorus to the ileocaecal junction and placed on paper towels soaked with isotonic saline over ice. The lumen of the intestine was washed out with 50 ml of ice-cold saline, and a small section from the proximal jejunum was removed and set aside for RNA isolation. The serosal surface was then patted dry, weighed, and opened along its length, and the mucosa was scraped off with a glass side. Care was taken to use the same amount of force along the entire length of the intestine and for each rat. The mucosa was then added to 50 ml of ice-cold saline and placed on ice while the remaining serosal tissue was weighed to allow for the calculation of wet mucosa mass. The mucosa was homogenised for 20 s at medium speed in a Heidolph Diax 600 homogenizer and then stored at 20°C.
Enzyme activities. Disaccharidase activity was assessed using the Dahlqvist method (7) with modifications. The assay system was confirmed to be linear with enzyme concentration.
Twenty-five microliters of appropriately diluted mucosal homogenate were added in duplicate to 25 µl of substrate solution containing 100 mM maleate buffer (pH 6.0), 56 mM sucrose or lactose, and 1% (vol/vol) toluene in a microtiter plate, sealed to prevent evaporation, and incubated at 37°C for 60 min. Three hundred microliters of Tris-glucose oxidase reagent containing 33 U/ml glucose oxidase, 500 mM Tris·HCl (pH 7.0), 0.01 mg/ml horseradish peroxidase, 0.1 mg/ml o-dianisidine, and 0.2% (vol/vol) Triton X-100 were added to halt the reaction and commence visualization of the produced glucose. The plate was sealed and incubated at 37°C for a further 60 min for color development. Absorbance of the plate was read immediately at 415 nm, and enzyme activity was calculated from the quantity of glucose produced after the dilution factor used was taken into account. One unit was defined as the ability to generate 1 µmol glucose/min, and results expressed as units per gram of wet mucosa mass. Wet mucosa mass was used to calculate specific activity rather than protein content, as protein concentration in the mucosa does not significantly change during iron deficiency (4, 5).
A subset of the disaccharidase assays were repeated using eight different concentrations of sucrose and lactose substrate solution diluted in 100 mM maleate buffer. A Lineweaver-Burk plot was constructed, and the Km value was calculated from the equation of the line.
IAP-II activity was determined by the method of Weiser (40). The assay system was confirmed to be linear with enzyme concentration. In brief, 10 µl of diluted mucosal homogenate were added in duplicate to 200 µl of substrate solution containing 500 mM Tris·HCl (pH 9.4), 10 mM MgCl2, 0.2 mM ZnCl2, and 0.23 mM di-tris-p-nitrophenylphosphate in a microtiter plate. The plate was then incubated at 37°C for 15 min, and the reaction stopped by the addition of 100 µl of 500 mM NaOH. Absorbance was measured at 415 nm, and activity was calculated from the quantity of nitrophenol produced after the dilution factor had been taken into account. One unit was defined as the ability to generate 1 µmol nitrophenol/min, and results were expressed as units per gram of wet mucosa mass.
Immunofluorescence.
Tissue segments 0.5 cm in length were removed from the proximal jejunum of a separate subgroup of iron-deficient and control rats, immediately frozen in liquid nitrogen-cooled isopentane, and stored at 80°C. Frozen sections between 8 and 10 µm thick were cut onto positively charged slides, air dried for 20 min, and then fixed in 4% formaldehyde in PBS for 5 min. Fixative was washed off with three 5-min washes with PBS.
Monoclonal antibodies against rat sucrase and lactase (BBC 1/35 and YBB 2/61, respectively) were obtained as a gift from Dr. Andrea Quaroni (29) and used as primary antibodies at a 1:250 dilution in PBS with 0.2% saponin. Samples were incubated with the primary antibody for 1 h before unbound antibody was removed by three 5-min washes with PBS. Anti-mouse IgG-FITC conjugate was used as the secondary antibody at a dilution of 1:250 in PBS with 0.2% saponin and incubated with the sample for 30 min before the unbound antibody was removed with three 5-min washes with PBS. Propidium iodide at a concentration of 0.05 µg/ml was added as a nuclear stain before the addition of a single drop of antifade mounting media, and the samples were then coverslipped. Slides were then viewed on a Biorad MRC1000 laser scanning confocal microscope.
Laser scanning confocal microscopy was not used as a quantitative measure of immunoreactive sucrase and lactase protein. Western blots were considered for this purpose; however, the antibodies used are unable to detect denatured protein.
Generation of radiolabeled probes.
Rat sucrase-isomaltase partial cDNA was obtained as a gift from Dr Susan Henning (6). Riboprobes were produced from the cDNA template using the Promega Riboprobe Combination Kit (SP6/T7) with [-35S]UTP (Amersham) as the radiolabel. The sucrase antisense probe was generated using T7 RNA polymerase. Probe specificity was confirmed by Northern blot analysis with hybridization to a single mRNA species at the correct weight range (data not shown).
In situ hybridization. In situ hybridization was performed as described previously (27). In brief, tissue from the proximal jejunum of the same rats used for immunofluorescence experiments was fixed in 4% paraformaldehyde, and wax sections were cut onto silanated slides. One million disintegrations/min of sucrase antisense riboprobe were applied to each slide and hybridized for 24 h at 55°C. Slides were then washed in 50% formamide buffer for 4 h before being washed for 30 min in 2x SSC and then for 30 min in 0.5x SSC at 65°C. Slides were then exposed to photographic emulsion (Amersham) for 60 h before development of the signal. Sections were counterstained with hematoxylin and eosin before being photographed on an Olympus BH-2 microscope fitted with a Sony DFW-SX900 digital video camera.
RNA isolation and generation of cDNA. RNA was obtained from the mucosa in the proximal jejunum of the same rats used for enzyme activities. Approximately 100 mg of tissue were homogenized in 1 ml of RNA-Wiz (Ambion) and isolated as per the manufacturer's instructions. Concentration and purity of the RNA was assessed by spectrophotometry. Ten micrograms of RNA were then treated with the Ambion DNA-Free kit to remove genomic DNA. Aliquots of DNA-free RNA were reverse transcribed with avain myeloblastosis virus reverse transcriptase and oligo(dT)15 primers according to the manufacturer's instructions (Promega). cDNA products were then purified with the UltraClean PCR Cleanup Kit (MoBio) and stored at 20°C for real-time PCR.
Real-time PCR. mRNA levels were determined by semiquantitative real-time PCR on a Lightcycler instrument (Roche). The PCR mix contained cDNA from an equivalent of 66 ng RNA, 0.55 µM of the appropriate forward and reverse primers, 500 µg/ml BSA, 1x Platinum SYBR green qPCR SuperMix UDG (Invitrogen), and additional MgCl2 (final concentration of 3.5 mM Mg2+) in a volume of 20 µl. The PCR program involved 2 min at 50°C for UDG PCR carryover decontamination, followed by an initial denaturation at 94°C for 2.5 min and then 50 amplification cycles of denaturing at 94°C for 30 s, annealing for 30 s, and extension for 60 s at 72°C. A touchdown method was used for annealing temperatures, which decreased from 70 to 56°C over 15 cycles. Primers were selected using Primer 3 software (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi), and the primer sequences used are shown in Table 1. Primer specificity was confirmed initially by PCR in a standard thermocycler and by sequencing the products using the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) at the Royal Perth Hospital (Perth, Australia). The consistency of target sequence produced by each sample in the Lightcycler was assessed by melting curve analysis and agarose gel electrophoresis where required.
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Data and statistics. All data are expressed as means ± SE. The Analyse-It General v1.71 software package was used for all computational statistics. One-way ANOVA was used to compare groups, with P < 0.05 considered significant.
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RESULTS |
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Gene expression.
Semiquantitative real-time PCR results are shown in Fig. 2. -Actin expression was not significantly different between the two conditions, validating the use of this gene as a measure of baseline cell function with respect to mRNA content in iron deficiency. Control rats exhibited significantly higher levels of both sucrase (1.8-fold) and lactase (3.1-fold) mRNA than iron-deficient rats, whereas PDX-1 expression was 4.5 times higher in iron-deficient rats. No significant change was observed for CDX-2, GATA-4, or HNF-1.
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DISCUSSION |
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On the basis of the decreased body mass of iron-deficient animals, alterations in sucrase and lactase activity may be attributable to decreased voluntary food intake in iron-deficient rats compared with control animals. Therefore, the use of a pair-fed control group should be considered. However, previous studies have reported significant reductions in disaccharidase activities at times when the food intake of iron-deficient rats was identical to controls, and alterations in body mass were not yet apparent (4, 5). In addition, it has been reported that protein-deficient diets increase disaccharidase activity, irrespective of iron status (31). As such, it seems that marginal macronutrient depletion plays little part in determining disaccharidase activity compared with the effects of iron deficiency, and thus pair-fed control experiments were not pursued.
To further examine the nature of the decrease in sucrase and lactase activities, the levels of jejunal gene expression were measured by performing semiquantitative real-time PCR for sucrase and lactase mRNA. Because results from these experiments show that iron-deficient rats also express the lowest levels of sucrase and lactase mRNA, it seems unlikely that alterations in disaccharidase activity in Wistar rats are caused by an inability to synthesize the enzymes but rather due to changes in gene expression. The lack of alteration in -actin expression suggests that iron deficiency induced this change through a specific and selective mechanism (see below).
To investigate potential mechanisms for the observed reductions in gene expression, we explored the possibility that sucrase and lactase protein expression may be restricted to a select region of the villus due to an alteration in enterocyte differentiation caused by iron deficiency. We investigated this possibility by performing confocal immunofluorescence microscopy for immunoreactive sucrase and lactase protein. The spatial pattern of sucrase and lactase protein expression observed along the crypt to villus axis is in full agreement with previous studies (6, 12, 33), but, more importantly, there does not appear to be any change resultant from iron status. Specifically, in both conditions, protein expression becomes apparent in enterocytes at the crypt-villus junction and along the length of the villus, with sucrase levels diminishing at the villus tip. Furthermore, the presence of sucrase and lactase protein exclusively on the microvillus membrane suggests that iron deficiency does not impair the posttranslational modification or targeting of the proteins.
The pattern of sucrase gene expression was also assessed by in situ hybridization. Consistent with immunofluorescence experiments, there was no discernable difference between the iron-deficient and control conditions. Performing in situ hybridizations for lactase was considered, but considering the similarities between overall gene expression and the pattern of protein expression the experiment was not pursued. Thus it seems unlikely that reductions in sucrase and lactase gene expression and, in turn, activity are due to any alteration in the state of proliferation and differentiation of the mucosa cells as they migrate from the crypt to the villus tip. It is more likely that a specific regulatory mechanism controls the abundance of sucrase and lactase mRNA in mature enterocytes and that this is sensitive to the effects of iron deficiency.
With respect to mechanisms that may regulate sucrase and lactase gene expression in response to iron deficiency, it seems unlikely that the common IRP/IRE system [for example, divalent metal transporter (DMT)-1, transferrin receptor, and ferritin mRNAs] is responsible for our observations. These systems operate relatively rapidly to regulate the abundance of mRNA and the resultant quantity of protein produced (18, 19, 32). By contrast, alterations in disaccharidase activity typically occur much more slowly, requiring weeks of iron supplementation to return to control values (9, 20). Analysis of the mRNA sequences of sucrase (NM_013061 [GenBank] ) and lactase (X56747 [GenBank] ) reveals three areas that have the C/CAGUG consensus sequence for the most common and physiologically relevant IRE bulge and loop structures (18, 19); these sequences can be seen at NM_013061 [GenBank] position 50135023 and X56747 [GenBank] positions 502512 and 59085918. However, only X56747 [GenBank] position 59085918 is in an untranslated region, and this sequence does not appear to contain sufficient matching base pairs to form a stable stem component, supporting the hypothesis that the IRP/IRE system has little control over the expression of these genes.
To further clarify potential mechanisms for the decrease in sucrase and lactase gene expression and the subsequent decrease in enzyme activity, we considered the possibility that mRNA levels are primarily regulated by the level of gene transcription. For this purpose, the expression of select transcription factors in the jejunum was measured by semiquantitative real-time PCR, including activators of the sucrase and lactase promoter regions CDX-2, GATA-4, and HNF-1 (3, 26, 37) as well as the potent repressor PDX-1 (15, 39). Strikingly, PDX-1 expression was 4.5-fold higher in the iron-deficient condition. Because any potential activation of the promoter regions by CDX-2 and HNF-1 appears to be entirely negated by PDX-1 (39), this strongly suggests that the decreases in sucrase and lactase gene expression are primarily due to a reduction of gene transcription.
Our observation that PDX-1 expression is elevated in the jejunum during iron deficiency has many wide-ranging implications, because there is a strong argument to suggest that PDX-1 is a major spatial regulator of proximal intestine gene transcription. PDX-1 expression is typically restricted to the pancreas and duodenum and is widely regarded as being vitally important for pancreatic development and function (1, 17, 22, 25, 28), whereas duodenal expression is required for adenosine deaminase promoter activation (8, 14). Furthermore, it has been hypothesized that the presence of PDX-1 is the primary reason sucrase and lactase expression is repressed in the duodenum (15, 39).
In this context, it is interesting to note that the expression of principal genes involved in the absorption of dietary iron, namely, DMT-1 and ferroportin (FPN)-1, are spatially restricted to the duodenum under normal circumstances (24, 35). Although the promoter regions for these genes have not yet been identified, it has been demonstrated that transcription of DMT-1 and FPN-1 is increased by iron deficiency in the human enterocyte cell line CaCo-2 (42). From a physiological standpoint, PDX-1 appears to be an excellent candidate transcription factor for the regulation of these genes (as has been established for adenosine deaminase), because increased PDX-1 expression in the jejunum may allow for reduced spatial restriction of gene expression and an increased capacity for iron absorption. Indeed, preliminary evidence suggests that both DMT-1 and FPN-1 expression is significantly increased in the proximal jejunum during iron deficiency, and this cannot be explained by IRE/IRP activity alone (A. R. West and P. S. Oates, unpublished observations). Potential mechanisms for the upregulation of jejunal PDX-1 during iron deficiency and the interaction with iron absorption proteins should therefore be explored in further studies.
In summary, the decrease in sucrase and lactase enzyme activities observed during iron deficiency appears to be primarily attributable to a significant reduction in gene expression. This reduction in expression is most likely caused by repression of sucrase and lactase promoter regions by PDX-1. The combined findings strongly support a role for PDX-1 in spatial regulation of numerous small intestine-related proteins and that this regulation is sensitive to the effects of iron deficiency.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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