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,3
* Graduate Center for Nutritional Sciences
Molecular and Cell Nutrition Laboratory, College of Agriculture, Departments of
Pediatrics
Cardiovascular Medicine, and
# Surgery, University of Kentucky, Lexington, KY 40546;
** Department of Food Sciences, University of Missouri-Columbia, Columbia, MO 65211; and
Department of Medicine, Vanderbilt University, Nashville, TN 37232
3To whom correspondence should be addressed. E-mail: bhennig{at}uky.edu.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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B and vascular cell adhesion molecule-1, were significantly increased in zinc-deficient mice compared with mice of the control or supplemented groups. In addition, zinc deficiency significantly reduced the DNA binding activity of peroxisome proliferator activate receptors (PPARs) in liver extracts. Interestingly, mRNA expression levels of PPAR
were significantly increased in thoracic aortae of zinc-deficient mice, indicating an adaptation process to decreased PPAR signaling. These data provide in vivo evidence of zinc deficiency inducing proinflammatory events in an atherogenic mouse model. These data also suggest that adequate zinc may be a critical component in protective PPAR signaling during atherosclerosis.
KEY WORDS: • zinc deficiency • atherosclerosis • inflammation • PPAR • lipoproteins
The development of atherosclerosis is usually due to multiple causes. Current patterns of energy intake frequently show the portion of the daily energy intake that is derived from fat to be well over 30% (1). Such nutritional habits were shown to result in hyperlipidemia and dyslipidemia. Much less attention has been paid to micronutrients, and particularly to minerals. However, epidemiologic studies suggest that in some population groups, low serum concentrations of zinc are associated with coronary artery disease (2). Furthermore, zinc concentrations were significantly lower in atherosclerotic plaques of abdominal aortae of deceased patients with ischemic heart disease and acute myocardial infarction (3). Mechanisms of the protective functions of zinc in the pathogenesis of atherosclerosis, including vascular cell dysfunction and the inflammatory response, are not clear.
We and others showed that zinc has antioxidant and anti-inflammatory properties (4–7). Zinc is a structural component of proteins that stabilizes thiol groups, thus rendering proteins less prone to oxidation (8). Oxidative stress can induce nuclear factor (NF)4 -B, a proinflammatory transcription factor involved in mediating endothelial cell activation [reviewed in (9)].
In addition to its antioxidant properties, we reported recently that zinc appears to be essential for the protective properties of peroxisome proliferator activated receptors (PPARs) 
and in vascular endothelial cells (10). PPARs are nuclear receptors that are involved in many metabolic pathways, including lipid and glucose metabolism (11). PPARs appear to have anti-inflammatory properties that can protect against endothelial cell activation (12,13). All of these features make PPARs attractive targets for pharmaceutical intervention in cardiovascular diseases. Indeed, clinical and experimental evidence suggests that PPAR activation decreases the incidence of cardiovascular diseases (11).
Zinc deficiency in atherosclerosis has not been studied in an animal model suitable for atherosclerosis research. LDL-receptor–deficient (LDL-R–/–) mice are a good model because, similar to humans, most cholesterol is carried in LDL (14). Here, we hypothesized that zinc deficiency can induce proatherogenic events such as oxidative stress and induction of inflammatory markers, and that zinc supplementation can reduce these oxidative stress and inflammatory markers. Furthermore, we aimed to determine whether the effect of zinc on inflammation was accompanied by modified PPAR activity.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Plasma cholesterol and lipoprotein profiles. Blood was drawn from exposed hearts using heparinized syringes. Plasma was obtained by centrifugation of whole blood at 1500 x g, 4°C, for 20 min. Plasma cholesterol concentration was determined enzymatically using a commercially available kit (Wako Chemicals).
The lipoprotein profile was determined by HPLC (Chemstation LC/MSD 1100, Agilent Technologies) (18). The chromatographic profile was monitored by a Diode Array Detector at 280 nm and a Mass Selective Detector (Agilent Technologies). Electrospray and chemical ionization were used to detect triacylglycerides and phospholipids, respectively (19). Proteins were detected by measuring absorbance at 280 nm.
Immunohistochemistry for vascular cell adhesion molecule-1 (VCAM-1) expression in aortic roots. Hearts were separated from thoracic aortae, cut horizontally, immersed in optimal cutting temperature embedding medium, and frozen at –20°C. Sections (10 µm) were cut on a cryostat (Microm HM505N, Carl Zeiss). Immunocytochemistry was performed as described previously (20). Briefly, endogenous peroxidase was inactivated using hydrogen peroxide (3%) in methanol. Samples were blocked in the serum of the secondary antibody host. Primary antibodies for VCAM-1 (PharMingen) were detected using biotinylated secondary antibodies and peroxidase ABC kits (Vectastain, Cat. # PK-4000). Aminoethylcarbozole was used as a chromogen, and sections were counterstained with hematoxylin.
Gene expression analysis.
Thoracic aortae were excised from mice and stored in RNAlater (Quiagen) until analysis. Total RNA was isolated from aortae using RNAeasy (Quiagen) after removing surrounding adipose and connective tissues. cDNA was generated using the Reverse Transcription System (Promega). Gene expression was determined by real-time PCR using the ABI Prism 7000 and Taq Man Universal PCR Master Mix (Applied Biosystems). Primers and probes were designed to VCAM-1 (gi: 31981429,forward 5' CGT CGC GAG GTT GTT TAG AGT 3',reverse 5' CAA CAG TCA GTC CAA GCA ACA CT 3',probe 5' 6-FAM CAG CAC TCC ATA TGC AGG TCA GAA CGT AAT C TAMRA 3'), PPAR (gi: 6755137, forward 5' CAC AAT GCC ATC AGG TTT GG 3', reverse 5' GCT GGT CGA TAT CAC TGG AGA TC 3', probe 5' 6-FAM ACA GGC CGA GAA GGA GAA GCT GTT GG TAMRA 3'), and glutathione reductase (gi: 34785373, forward 5' CGA CCA TGA TTC CAG ATG TTG A 3', reverse 5' TCA GAT TCA GGC CCT TAG AAT TTG 3', probe 5' 6-FAM TGC CTG CTC TGG GCC ATT CGA C/36-TAMRA 3') using the Primer Express 2.0 software (Applied Biosystems) and synthesized by IDT DNA. Detection of 18S RNA utilized predeveloped assay reagents (Applied Biosystems). Relative quantifications employed standard curves for individual genes using serial dilutions of RNA samples expected to show induced gene expression, i.e., a random sample from the zinc-deficient group for VCAM-1 and a random sample from the zinc-supplemented group for glutathione reductase and PPAR gene expression studies.
Transcription factor (NF-B and PPAR) activation studies: electrophoretic mobility shift assay.
Left liver lobes were flash frozen and stored at –80°C until analysis. Nuclear extracts containing active proteins were prepared according to the method of Dignam et al. (21). Nuclear extracts were incubated for 25 min with 32P-end-labeled oligonucleotide probes containing the enhancer DNA element for NF-B (Promega) or the PPAR response element (PPRE) (Santa Cruz). Incubation at room temperature was performed in the presence of nonspecific competitor DNA. After binding, the complexed and uncomplexed DNA in the mixture was resolved by electrophoresis in a 6.5% (wt:v) nondenaturing polyacrylamide gel and visualized by autoradiography. Control reactions using a supershift assay were performed to demonstrate the specificity of the shifted DNA-protein complexes. The antibodies used were obtained from Santa Cruz.
Quantifications and statistical analyses. Samples of all mice (n = 11) were subjected to analyses of zinc concentrations, plasma lipoproteins, mRNA expression, and transcription factor binding activities. VCAM-1 staining in aortic roots took place in 5 mice/treatment group. Numeric data were analyzed using SYSTAT 7.0. Comparisons between treatments were made by 1-way ANOVA with post-hoc comparisons of the means made by Tukey’s least significance difference procedure. Differences with P < 0.05 were considered significant. Photomicrographs of VCAM-1 staining in aortic roots were analyzed using a double-blind design. Uncropped pictures of the same magnification were assigned random labels. Staining intensity of photomicrographs was then evaluated by 5 individuals by ranking the specimen according to staining intensity.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Plasma total cholesterol and lipoprotein concentrations of triacylglycerides, cholesterol, and phospholipids. Although the LDL fraction was not affected, free cholesterol was significantly reduced by zinc supplementation in the VLDL and HDL fractions compared with the zinc-deficient mice (Table 2). Cholesterol esters were significantly higher in VLDLs and LDLs of zinc-deficient mice compared with the 2 other groups (Table 2). Triacylglycerides differed significantly in VLDLs among all groups, with the lowest concentrations in zinc-supplemented mice. In addition, total cholesterol, as determined enzymatically, was modified by zinc. Zinc-supplemented mice had significantly lower (4.24 ± 0.25 mmol/L) total plasma cholesterol concentrations than zinc-deficient mice (5.22 ± 0.33 mmol/L). Control mice had cholesterol values (4.59 ± 0.26 mmol/L) that did not differ from those of the other groups. Triacylglycerides in the LDL fraction also were significantly different in zinc-supplemented mice compared with the other 2 groups (Table 2). Phospholipids did not differ among treatment groups in the LDL fraction but were significantly different in the VLDL and HDL fractions of zinc-supplemented compared with zinc-deficient mice, with the highest concentrations in zinc-deficient mice (Table 2).
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Liver transcription factor activities.
Zinc deficiency significantly increased the DNA binding activity of the proinflammatory transcription factor NF-B in liver tissues (Table 3). Control and zinc-supplemented mice did not differ in DNA binding activity of this transcription factor. On the other hand, the anti-inflammatory transcription factor PPAR was most activated in the livers of control mice, and the lowest DNA binding levels occurred in zinc-deficient mice (Table 3).
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mRNA expression.
PPAR mRNA was significantly greater in thoracic aortae of mice fed the zinc-deficient diet than in those of mice fed the control diet (Table 3). PPAR mRNA expression values from mice in the zinc-supplemented group did not differ from those of the other groups. |
DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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As expected, femur zinc was significantly modified by both zinc deficiency and supplementation. On the other hand, we did not detect changes in the liver zinc concentrations, indicating a mobilization of zinc stored in bone to replenish soft tissue zinc pools.
The effect of zinc on plasma lipid concentrations remains a controversial subject. Other investigators studying different model systems found increased concentrations of cholesterol, phospholipids, VLDL, and intermediate density lipoproteins during zinc deficiency (24). On the other hand, it was reported that zinc deficiency decreased plasma and VLDL triacylglyceride concentrations in rats (25). However, this effect was attributed mainly to the lower food intake resulting from zinc deficiency. The effect of zinc deficiency has never been studied in a mouse model appropriate for studying atherosclerosis. In this study, we utilized LDL-R–/– mice, which have a lipoprotein profile similar to that of humans (14). Here, we observed a striking difference in cholesterol, triacylglycerides, and phospholipids in the lipoprotein fractions between zinc-deficient and zinc-supplemented mice. The lipid-lowering effect of zinc was also shown in humans (26). The mechanism for the effect of zinc on lipoproteins is not clear. It was suggested that zinc is required for enzymes involved in lipid synthesis and lipoprotein excretion (27). For example, Porsch-Ozcurumez et al. (28) reported that zinc finger protein 202 (ZNF202) inhibited the expression of ATP-binding cassette transporter A1 and G1, both responsible for the efflux of cholesterol and phospholipids.
The upregulated expression of glutathione reductase in the thoracic aortae of zinc-deficient mice could be the result of increased oxidative stress with resulting upregulation of genes involved in the defense against free radicals. Furthermore, we observed increased plasma isoprostane concentrations in mice fed a zinc-deficient diet, also indicating an increase in oxidative stress due to zinc deficiency (data not shown). Isoprostanes are prostaglandin-like compounds that are formed from the peroxidation of arachidonic acid; they can be used as reliable and quantifiable markers of oxidative stress (29).
Oxidative stress can induce inflammation and contribute to the progression of atherosclerosis. We and others reported previously that inflammatory pathways, such as the NF-B pathway, are sensitive to oxidative stress. We showed in endothelial cell cultures that zinc deficiency can induce NF-B DNA binding activity [reviewed in (30)]. Here, we provide in vivo evidence for the zinc deficiency–mediated induction of NF-B activity in liver extracts. NF-B has a key role in the transcription of inflammatory genes that induce endothelial cell dysfunction and thus mediate monocyte recruitment. VCAM-1 is a very important example of a NF-B target gene that is involved in leukocyte attachment to the endothelial cells and diapedesis through the endothelium (31). The fact that VCAM-1 mRNA and protein expression were upregulated in aortic tissues of mice fed a zinc-deficient diet indicates that NF-B was most likely activated not only in livers but also in the vasculature. In fact, we previously observed a marked induction of NF-B in cultured endothelial cells during zinc deficiency (32).
NF-B activation and VCAM-1 expression were reported to be inhibited by PPAR and agonists (12,13). PPARs are nuclear receptors expressed in a wide variety of tissues, including vascular endothelial cells (13). We reported recently that zinc is required for the anti-inflammatory properties of PPAR and agonists against tumor necrosis factor–induced endothelial cell activation (10). In the current study, we showed that PPAR binding activity in liver extracts can be modified by nutritional zinc intake. Because zinc concentrations in liver tissues did not change in response to various amounts of dietary zinc, the link between zinc and PPAR DNA binding activity remains unresolved. Previous data obtained from cell culture studies, however, showed that PPAR and agonists could not induce PPAR binding activity in zinc-deficient endothelial cells (10).
We have provided in vivo evidence that zinc deficiency induces atherosclerotic events in LDL-R–/– mice. We showed a significant upregulation of VCAM-1 in aortic tissues and increased plasma lipids in zinc-deficient mice. Furthermore, zinc deficiency appeared to induce oxidative stress and modify PPAR expression and activity. Zinc supplementation decreased plasma lipids below control levels but did not significantly improve other markers compared with the control diet. Because dietary zinc intake of certain population groups is still below intake recommendations (22), these data underline the importance of zinc for the prevention of cardiovascular diseases such as atherosclerosis.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Present address: Department of Food Science and Human Nutrition, Iowa State University, Ames, IA 50011.
4 Abbreviations used: LDL-R–/–, LDL-receptor–deficient; NF, nuclear factor; PPAR, peroxisome proliferator activated receptor; PPRE, PPAR response element; VCAM-1, vascular cell adhesion molecule-1.
Manuscript received 16 December 2004. Initial review completed 11 February 2005. Revision accepted 23 May 2005.
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