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Nutrient-Gene Interactions

Transcriptome and Proteome Analysis Identifies the Pathways That Increase Hepatic Lipid Accumulation in Zinc-Deficient Rats^1,2

Heike tom Dieck^*, Frank Döring, Dagmar Fuchs^**, Hans-Peter Roth^** and Hannelore Daniel^**,3

^* Degussa Food Ingredients GmbH, Lise-Meitner-Strasse 34, 85354 Freising, Germany; University of Kiel, Research Group Molecular Nutrition, Hermann-Weigmann-Strasse 1, 24103 Kiel, Germany; and ^** Technical University of Munich, Molecular Nutrition Unit, Hochfeldweg 2, 85350 Freising-Weihenstephan, Germany

³To whom correspondence should be addressed. E-mail: Daniel{at}wzw.tum.de.

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
For identification of the underlying molecular changes in hepatic lipid metabolism in zinc deficiency, rats were force-fed a zinc-deficient diet. Subsequently DNA-microarray and proteome profiling was performed in combination with hepatic lipid analysis. Of 6200 target sequences analyzed, 268 transcripts showed altered expression levels in livers of zinc-deficient rats, with 43 genes thereof related to hepatic lipid metabolism. Northern blot analysis and quantitative real-time RT-PCR were employed to confirm changes in mRNA levels. Proteins involved in lipid metabolism were identified by proteome analysis. Functional gene clusters with uniform changes in transcript levels suggested that the pathways required for lipolysis and mitochondrial as well as peroxisomal fatty acid degradation were downregulated, whereas those needed for de novo fatty acid synthesis and triglyceride assembly were increased. Subsequent enzymatic analysis of liver tissues confirmed an almost 40% greater triacylglycerol concentration in zinc-depleted rats, as well as an altered fatty acid composition of the lipid fraction as determined by gas chromatography. Liver lipids of zinc-deficient rats had significantly greater proportions of cis-9-oleic acid, cis-11-vaccenic acid, caprylic acid, myristic acid, -linolenic acid, and eicosapentaenoic acid, and significantly less stearic and arachidonic acids. These alterations in hepatic metabolism are discussed in the context of changes in mRNA and protein levels of enzymes and transporters responsible for fatty acid metabolism, sequestration, and their transcriptional control.

KEY WORDS: • zinc deficiency • lipid metabolism • gene expression • DNA array • PPAR

Zinc is a constituent of hundreds of proteins with catalytic and structural roles and is involved in intermediate metabolism, hormone secretion pathways, and immune defense (1–3). It is also an essential component of many transcription factors, suggesting that alterations in zinc status are immediately translated into changes in gene expression (4,5). A number of mRNA species, for example, of metallothionein, cholecystokinin, uroguanylin, endothelin, p53, retinol binding protein, and apolipoproteins, have been shown to be either increased or decreased in expression as a consequence of changes in zinc status (6–12). The phenotypical metabolic alterations caused by zinc deficiency have been established in both humans and experimental animals and result in anorexia, impaired immunity, skin lesions, abnormal development, and growth retardation (13–15). Previous studies also reported major alterations in lipid metabolism in zinc-deficient rats. Besides changed lipoprotein formation, changes in the fatty acid composition of lipids in different organs during zinc deficiency have also been reported (10,16–20). Because zinc-deficiency symptoms very much resemble those of an essential fatty acid deficiency, a close link between fatty acid metabolism and zinc status was proposed (18,21,22). Moreover, an increased triacylglycerol accumulation in liver of zinc-depleted rats has been described and, depending on the dietary fat component, rats developed fatty livers (22,23). Increased activities of some lipogenic enzymes and decreased activities of those needed for fatty acid degradation caused by zinc deficiency have been demonstrated (24). The underlying mechanisms for these pronounced changes in hepatic metabolism are unknown and, strikingly, none of the lipogenic/lipolytic enzymes is known to contain zinc as an essential constituent. This work is a follow-up to a previous gene expression analysis of around 1000 target sequences of hepatic metabolism in zinc-deficient rats (25). We here employed an oligonucleotide microarray with >5000 targets genes for assessing changes in the transcriptome in combination with proteome analysis as global screening methods and analysis of key lipid metabolites. All analytical variables were obtained from the same samples to determine changes in mRNA and protein steady-state levels that could be linked to individual pathways of liver metabolism that change triglyceride content and fatty acid composition in hepatic lipids. Our analysis demonstrates that the array and proteome technologies are helpful in identifying regulated gene/protein clusters that functionally resemble individual pathways of hepatic carbohydrate and lipid metabolism and that show uniform changes.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
    Animals, diets, and zinc status. Zinc deficiency in rats was achieved by force-feeding as described recently (25). In brief, 24 male rats with a mean body weight of 122.6 ± 4.8 g were randomly divided into 2 groups. All rats received a purified diet (AIN 93G) as described by Kraus et al. (26) with casein depleted of zinc as the protein source. The dietary fat was corn oil with the following fatty acid content (% w:w): palmitic acid, 10.5; oleic acid, 28.5; linolenic acid, 1.3; stearic acid, 1.7; and linoleic acid, 56.6. One group of rats was fed a zinc-adequate diet (382 µmol Zn/kg diet), and the other group received the zinc-deficient diet (19.8 µmol Zn/kg diet). For synchronization of food intake, all rats were force-fed by gastric tube 4 times per day (0800, 1200, 1800, and 2200 h) as described (26). Rats were weighed every day. After 11 d of feeding, the rats were anesthetized with diethyl ether and decapitated. Blood was collected in heparinized tubes. Resected tissues were immediately frozen at –196°C and stored at –80°C. Zinc status was assessed by determination of serum zinc concentration and activity of serum alkaline phosphatase (EC 3.1.3.1) as described elsewhere (27,28). The liver zinc concentration was measured by atomic absorption spectroscopy (Model No. 5100, Perkin-Elmer) as described (26).

    Pan® 5K Pan rat oligonucleotide array. In addition to the array experiments described earlier (25), a new analysis from the same liver samples employing an oligonucleotide array with 5535 gene-specific oligonucleotides probes (50 mer) obtained from MWG Biotech was conducted. Probing of arrays was performed according to the manufacturer’s recommendations.

In brief, for the oligonucleotide array analysis, 100 µg total RNA was reverse transcribed in the presence of either Cy3- or Cy5-labeled dCTP (Amersham Bioscience Europe) to produce fluorescence-labeled first-strand cDNAs. Samples from livers of control rats (n = 3) were labeled with Cy5. Samples from livers of zinc-deficient rats (n = 3) were labeled with Cy3. Arrays were scanned (Affymetrix 428 Array Scanner) under dried conditions. The data obtained were normalized and analyzed using ImaGene^TM 4.2-Software (BioDiscovery). Three independent hybridizations were carried out. Genes were considered up- or downregulated if the change was 1.6-fold or greater in at least 2 hybridizations. This arbitrary threshold was chosen because, in most cases, Northern blot analysis revealed similar or even greater changes in transcript levels than that detected by array analysis.

    Northern blots and quantitative real-time RT-PCR. Because array data frequently underestimate the alterations in transcript levels (29), a subset of genes for which changes in the microarray screening were observed was analyzed by Northern blot analysis or real-time RT-PCR. Northern blot analysis was carried out using an established capillary blotting method (30) as described (25). Ten micrograms of total-RNA/lane was separated on denaturing formaldehyde for subsequent blotting. cDNA fragments representing unique open reading frames of the 2 genes were used for hybridization whereas other genes have been confirmed in regulation already in our previous analysis (25): glycerol kinase (J00750, 124–366) hydroxy-phythanoyl-lyase (AJ245707, 103–262). cDNA fragments were randomly labeled with [ ³²P]-dATP (ICN Biomedicals). Hybridization signals were visualized by exposure to a phosphorimager screen. Imager screens were scanned depending on signal intensity after 24 to 96 h of exposure. After being stripped, blots were reprobed for glyceraldehyde-3-phosphate dehydrogenase (GAPDH, Accession No. M17701, nucleotides 549-1001) for normalization. cDNA fragments representing unique open reading frames cytochome P450 (Cyp)⁴ VIA3 (FP, ATG GAG GAT GGG AAG AGC TTG; BP, TAG CTT CTT GAG ACG CAG CTG), carboxylesterase (FP, GTG TTA TCT CCT CTG GCC AAG; BP, ACC ATG GTC TCC GAT CAC TGT), and S14 (FP, CTA CTG AAG GTC ATG GAT CGG; BP, GGT AAG GAT GTG ATG GAG GCT) were used for primer design for quantitative real-time RT-PCR (LightCycler, Roche). For online detection during real-time PCR analysis the fluorescent dye SYBR Green from Fast start SYBRGreen (Roche Diagnostics) was used. Melting curves and native gel electrophoresis were used to characterize the final products.

    Sample preparation for 2D-PAGE. Sample preparation was carried out as described elsewhere (31). In brief, frozen liver was ground in liquid nitrogen. Powdered tissue was weighed, 200 µg lysis buffer (31) per 10 mg tissue was added, and the tissue was homogenized by ultrasonification (30 strokes, medium amplitude) on ice. The lysate was centrifuged for 30 min at 1,000,000 x g at 4°C and the supernatant containing the solubilized proteins was used directly or stored at –80°C.

    2D-PAGE. 2D-PAGE, isoelectric focusing (IEF) in the first dimension and SDS-PAGE in the second dimension, was carried out as described by Herzog et al. (31). Separation in the first dimension was performed on 18-cm IPG strips using an Amersham IPGPhor unit (pH 4–7 and 6–11). IEF was carried out under the following conditions: 500 V (10 min, gradient), 4000 V (1.5 h, gradient), 8000 V (25,000 Vh, Step-n-hold). Subsequently to IEF, strips were re-equilibrated before being loaded onto SDS-PAGE gels. SDS-PAGE gels were casted according to the method of Laemmli and were run using an Amersham Biosciences Ettan-Dalt II system employing the following conditions: 4 mA per gel for 1 h and then 12 mA per gel. Gels were then stained overnight in Coomassie solution and destaining occurred in aqua bidest until the background was completely clear (31).

    Analysis of gels. The Coomassie-stained gels were scanned using an Image Scanner (Amersham Biosciences) and the spots were detected using ImageMaster Software (Amersham Biosciences) including background subtraction and volume normalization. Gels were matched to a reference gel (virtual gel containing all spots detected on any gel) and then average gels derived from control or zinc-deficient rats were generated. Gels from at least 3 independent protein samples of liver tissue of control and zinc-deficient rats were compared to each other and those spots that differed at least 2-fold were chosen for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis.

    Enzymatic digestion of proteins spots for MALDI-TOF-MS. MALDI-TOF-MS analysis was performed as described (31). In brief, Coomassie-stained spots were destained with alternating washing procedures until the stain was completely removed. After trypsin digestion, the peptide fragments were extracted and stored. A second extraction was carried out under identical conditions and supernatants derived from a spot were collected and analyzed.

    MALDI-TOF-MS anaylsis of tryptic peptides. Mass analysis was carried out according to the method of Bruker Daltonics using the Autoflex mass spectrometer of Bruker Daltonics as described earlier (31). The proteins were identified by the use of the Mascot Server (Bruker Daltonics) based on mass searches within rat sequences only. The search variables allowed for the carboxyamidomethylation of proteins. A minimum score of 63 and a mass accuracy of ±0.1% were set, and repeated (at least 2 times) identification of the same fragments of 2 independent gels was required. The protein also had to exhibit a significant difference in the number of matched peptides to the next potential hit.

    Enzymatic determination of triacylglycerol concentration in liver. The liver lipid fraction was extracted with an hexane-isopropanol mixture (3:2, v:v, containing 50 mg BHT as antioxidant) as described by Hara and Radin (32) and determined quantitatively by a gravimetric method. For analysis of triacylglycerols, the extract was dissolved in Triton X-100 as described by de Hoff et al. (33). Total triacylglycerol concentrations were measured using the enzymatic reagent kit Ecoline 25 obtained from Merck.

    Gas chromatography. Fatty acids of liver lipids were converted into the corresponding methylesters (FAME) with the derivation reagent TMSH. FAMEs were separated by gas chromatography using a Hewlett-Packard 6850 gaschromatographic system fitted with programmed temperature vaporizing split/splitless injector, autosampler (6850 Automatic Liquid Sampler, Agilent Technologies), flame ionization detector, and a 30-m DB-23 [(50%-cyanopropyl)methylpolysiloxane] capillary column (diameter = 250 µm, layer thickness = 0.25 µm). FAMEs were identified by comparing retention time with that of individual methyl esters of 22 standard fatty acids. The following fatty acids (Sigma) served as standards: 6:0 (capronic), 8:0 (caprylic), 10:0 (capric), 12:0 (lauric), 14:0 (myristic), 14:1 (myristoleic), 15:0 (pentadecanoic), 16:0 (palmitic), 18:0 (stearic), 18:1 (cis-9-oleic), 18:1 (cis-11-vaccenic), 18:2 (linoleaidic), 18:2 (linoleic), 18:3 (-linolenic), 18:3 (-linolenic), 20:0 (arachidic), 20:1 (eicosenoic), 20:3 (eicostrienoic), 20:4 (arachidonic), 20:5 (eicospentaenoic), 22:4 (docosatetraenoic), 22:6 (docosahexaenoic). Quantification was performed based on AUC as a percentage of the total fatty acid amount (total AUC).

    Statistical methods. Calculations were performed by using Prism 2.01 (GraphPad Software) and Microsoft Excel (Windows 2000, Microsoft). For each variable, 3 to 8 independent experiments were carried out. Differences between diet groups at d 11 were tested for significance using an unpaired Student t test. Differences were considered significant at P 0.05.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
    Zinc status. Zinc-deficient rats had a significantly reduced weight gain (–28%), reduced zinc concentrations in serum (–58%) and liver (–24%), reduced activity of serum alkaline phosphatase (–37%), and almost undetectable hepatic metallothionein-1-mRNA as reported earlier (25). In addition, rats fed the zinc-deficient diet displayed typical symptoms of zinc depletion such as spars and rough hair as well as mild skin lesions.

    Identification of genes encoding proteins involved in hepatic lipid metabolism with altered expression. Cross analysis of the acquired expression data sets showed that altogether 4100 (66%) of the total 6200 different genes spotted signal intensities above background levels could be detected (data not shown). Within the chosen arbitrary threshold, 268 genes were identified as candidate genes of zinc regulation (data not shown). Thereof, 43 could be identified as carrying a biological function in hepatic lipid metabolism (Table 1). This cluster of genes included 9 mRNA species with increased and 34 with reduced steady-state expressions, including those encoding key enzymes of hepatic lipid metabolism such as acyl-CoA oxidase, acyl-CoA dehydrogenase, ATP-citrate lyase, and others as well as transcriptional regulators such as the peroxisome proliferator activated receptor, PPAR-. A graphical allocation of the identified transcripts according to the function of the corresponding proteins would demonstrate that zinc deficiency causes cytoplasmatic as well as mitochondrial and peroxisomal pathways of hepatic lipid metabolism to change.

The fatty acid analysis showed 2 fractions with 12 different fatty acids each representing >1% of the total fraction (main fraction) and 21 fatty acids as the minor fatty acid fraction with each representing <1% of total fatty acid fraction. Thereof, 5 fatty acids of the main fraction and 7 of the minor fraction showed significant alterations in abundance in livers of zinc-deficient rats (Table 4). The most prominent changes included significant reductions in stearic and arachidonic acid contents, whereas those of cis-9-oleic, cis-11-vaccenic, caprylic, myristic acid, -linolenic, and eicosapentaenoic acid were significantly elevated in livers of zinc-deficient rats.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

We here report significantly altered expression levels of a large number of genes coding for key enzymes of hepatic lipid metabolism with as many as 37 transcripts linked directly to triglyceride turnover. We also observed a markedly increased triglyceride accumulation in liver tissue of zinc-deficient rats as reported previously in force-fed zinc-deficient rats (22). When the hepatic genes/proteins identified as differentially regulated by zinc deficiency are arranged in the context of their biological functions, it becomes obvious that enzymes required for hepatic triacylglycerol turnover and ß-oxidation of fatty acids uniquely showed reduced mRNA steady-state levels, whereas those of de novo lipogenesis displayed increased levels. Although not all genes encoding proteins of these pathways were found on the array, altered transcript levels of a variety of enzymes as well as hepatic factor S14 argued for major changes in these metabolic routes. A functional correlate of these changes is the increased activities of key enzymes of the fatty acid synthesis pathway such as acetyl-CoA carboxylase and fatty acid synthase, as observed previously in the same animal model (24). The higher hepatic triglyceride concentration may result not only from an increased de novo fatty acid synthesis and triglyceride assembly, but also from a simultaneously decreased breakdown or release of triglycerides from liver (22). An increased fat accumulation was also found in intestinal cells with a reduced export of triglycerides and impaired chylomicron assembly in zinc-deficient rats (34). In liver we observed reduced expression of the carboxylesterase gene. This enzyme is the functional equivalent of the hormone-sensitive lipase (37) and could cause a decreased flow of fatty acids from hepatic triglyceride stores to the place of VLDL assembly (38), thereby reducing hepatic triglyceride output.

Many of the symptoms of zinc deficiency resemble those of an essential fatty acid deficiency, and zinc deficiency seems to intensify the symptoms of the latter (18). A change in tissue fatty acid composition depending on zinc status has been reported for a variety of tissues (16,17,19) with major differences in concentrations of oleic, linoleic, and arachidonic acid (16,19,20,39) and in the ratio of saturated and monounsaturated to polyunsaturated fatty acids (22). Zinc was postulated to directly affect desaturation processes (16,17,19), although changes of activities of 5-, 6-, and 9-desaturases were not consistently found in zinc-deficient rats (23,24,39). We observed a substantially reduced arachidonic acid concentration and a modest increase in linoleic acid concentration in liver similar to a previous study in rats force-fed a zinc-deficient diet (22).

Although mRNA expression of desaturase genes could be detected, their steady-state levels did not change significantly in zinc deficiency, which was in accordance with unchanged -5 and -6 desaturase activities in zinc-deficient rats in the same model (23,36). However, various enzymes involved in fatty acid degradation and conversion such as CYP4- and CYP2C-isoenzymes (CYP4A3, CYP2C23), which as hydroxylases are involved in metabolism of saturated and unsaturated fatty acids (40), and the 3,2-enoyl-CoA isomerase (ECI) did show substantially reduced transcript levels. The rat ECI is an auxiliary enzyme involved in peroxisomal ß-oxidation of unsaturated fatty acids and is essential for the breakdown of oleic acid (41). Although elevated concentrations of oleic acid in zinc deficiency were thought to result from an elevated 9-desaturase activity (19,39), our data suggest that oleic acid degradation may be impaired by the lower ECI level. Because all transcripts of desaturation pathways appeared unchanged, the altered fatty acid composition of hepatic lipids in zinc-deficient rats most likely reflects the downregulation of enzymes required for fatty acid oxidation in peroxisomes and mitochondria (Tables 1, and 3) including L-3-OH acyl CoA dehydrogenases with a specificity for short-chain fatty acids. In addition, the fatty acid import system in the inner mitochondrial membrane represented by the carnitine acyltransferases showed reduced expression levels. Therefore, it is conceivable to assume that import and oxidation of fatty acids by mitochondrial pathways and the peroxisomal breakdown as well may be reduced. This could explain the markedly increased levels of fatty acids and in particular those with short and medium chain length.

How can these pleiotropic effects of zinc deficiency on hepatic lipid metabolism be explained? PPAR-, a member of the zinc-containing superfamily of nuclear receptors of the thyroid/steroid hormone super class, as well as sterol regulatory element binding protein (SREBP-1), is known as the most important transcriptional regulator of genes involved in lipid metabolism (42). PPARs contain in the DNA binding domain at least 2 of the characteristic zinc finger domains as structural elements essential for proper function of the proteins (43). After activation, PPARs bind to specific receptor sites within the specific DNA "response element" (PPRE) for transcriptional activation of target genes in heterodimeric complexes mainly with 9-cis-retinoic acid X receptor (RXR) and additional nuclear coactivators and corepressors. In liver, PPAR- potently regulates in concert with counteracting regulators such as SREBP mitochondrial and peroxisomal fatty acid utilization pathways (44,45) as well as triglyceride synthesis and release (46).

Among many other targets, PPAR- alters cytochrome P450 expression (47,48) with most of the P450 enzymes also involved in -oxidation of certain fatty acids and cellular stress response. Inspection of the regulated transcripts in liver of zinc-deficient rats (Table 1) reveals a huge set of genes for which a PPAR--mediated transcriptional control has been reported (44,49–53). Although this may just be coincidental, almost all identified genes with a prime role of PPAR- in expression control do show an opposite regulation in zinc deficiency (25) and, moreover, a decline in PPAR- mRNA. This suggests that most or even all of PPAR--mediated processes could simultaneously be impaired by zinc deficiency, even in the presence of a functional SREBP pathway. It may be speculated that the essentiality of zinc as a structural element in this key transcriptional regulator causes these uniform changes in hepatic lipid and P450 xenobiotic metabolism. This assumption appears to be supported by a recent study on the effect of zinc chelators on PPAR- expression, signaling, and activity, which showed a reduction upon zinc chelation and reconstitution after zinc addition (54).

Another member of the zinc finger transcription factor superfamily is the thyroid receptor TR. Zinc deficiency was shown to alter the biological effects of thyroid hormones but the mechanism by which the zinc status changes thyroid hormone functions is not resolved (55). An impaired signaling of T3 in zinc deficiency via a diminished receptor binding and DNA interaction and a decreased expression rate of downstream genes has been reported (56). In support of these observations, we identified a variety of target genes of T3 such as carnitine palmitoyltransferase I (Table 1), IGFBP-1, and IGFBP-2 (25) with reduced transcript levels. Interestingly, proteome analysis also revealed a reduced level of liver thyroid hormone receptor ß, which could be part of the impaired T3 signaling cascade (Table 3). One of the key T3-dependent hepatic genes encodes the lipogenic factor S14 that surprisingly displayed increased mRNA levels in zinc deficiency. However, S14 gene transcription is regulated in a complex manner and is known to be affected by ligands of PPAR as well as by SREBP pathways. A cho-response element in the promotor provides an additional link to ligands from carbohydrate metabolism (57,58). Moreover, TR as well as PPAR- does form heterodimers with RXR and both receptors are reciprocally inhibited in interaction with RXR (59). Any change in either activation process (T3 or PPAR-) at the level of receptor dimerization or by the requirement of zinc for zinc finger-dependent transcriptional control could alter the integrated response in common target genes such as S14. Assuming an impaired PPAR--dependent transcription control in livers during zinc deficiency, the inhibition of peroxisomal proliferators on S14 expression would be reduced and become independent of zinc but could be enhanced via SREBP, a stimulator of S14 transcription (60). Whereas for T3 action, the TR receptor still could retain its ability for T3 binding, the zinc-dependent interaction with the promoter sequence may be blunted (58), in particular because ligand-activated TR would compete with PPAR- for interaction with the dimerization partner RXR. As an integrated output of this impaired expression control, hepatic S14 levels could be elevated in zinc deficiency. The central paradigm of how zinc deficiency may cause alterations in expression of many of the hepatic genes/gene clusters in relation to PPAR- and SREBP-activated processes is summarized (Fig. 1). Our hypothesis of course requires further studies for substantiation, for example, in a PPAR- null mouse model subjected to experimental zinc deficiency.

View larger version (27K): [in this window] [in a new window]   FIGURE 1 Postulated mechanisms by which zinc deficiency may alter gene expression of proteins in hepatic lipid metabolism [modified as in Ref. (61)]. TR, thyroid receptor.

	FOOTNOTES

 
¹ Poster presented at the First International Nutrigenomics Conference in Noordwijk aan Zee (Netherlands) 28 February 2001. March 2002 [tom Dieck, H., Döring, F., Roth, H. P., Pfaffl, M. & Daniel, H. Identification of genes with altered expression in zinc-deficient rats by use of DNA microarrays].

² Partially supported by Degussa Food Ingredients GmbH, Freising, Germany.

⁴ Abbreviations used: CYP, cytochrome P450; ECI, 3,2-enoyl-CoA isomerase; IEF, isoelectric focusing; MALDI-TOF, matrix assisted laser desorption/ionization time of flight; PPAR, peroxisome proliferators activated receptor, PPRE, PPAR response element; RXR, 9-cis-retinoic acid X receptor; SREBP, sterol regulatory element binding protein; Zn, zinc; +Zn, zinc-adequate, –Zn, zinc-deficient.

Manuscript received 21 June 2004. Initial review completed 18 August 2004. Revision accepted 15 November 2004.

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