Plasma zinc's alter ego is a low-molecular-weight humoral factor.

Mild dietary zinc deprivation in humans and rodents has little effect on blood plasma zinc levels, and yet cellular consequences of zinc depletion can be detected in vascular and other tissues. We proposed that a zinc-regulated humoral factor might mediate the effects of zinc deprivation. Using a novel approach, primary rat vascular smooth muscle cells (VSMCs) were treated with plasma from zinc-deficient (<1 mg Zn/kg) or zinc-adequate (35 mg Zn/kg, pair-fed) adult male rats, and zinc levels were manipulated to distinguish direct and indirect effects of plasma zinc. Gene expression changes were analyzed by microarray and qPCR, and incubation of VSMCs with blood plasma from zinc-deficient rats strongly changed the expression of >2500 genes, compared to incubation of cells with zinc-adequate rat plasma. We demonstrated that this effect was caused by a low-molecular-weight (∼2-kDa) zinc-regulated humoral factor but that changes in gene expression were mostly reversed by adding zinc back to zinc-deficient plasma. Strongly regulated genes were overrepresented in pathways associated with immune function and development. We conclude that zinc deficiency induces the production of a low-molecular-weight humoral factor whose influence on VSMC gene expression is blocked by plasma zinc. This factor is therefore under dual control by zinc.

Suboptimal dietary zinc intake promotes vascular inflammation and atherogenesis in a mouse model of atherosclerosis.

SCOPE: Cardiovascular health is strongly influenced by diet. Zinc has antioxidant and anti-inflammatory properties but its long-term influence on vascular health at dietary intake levels relevant to the human population in developed countries has not been studied. We investigated the influence of suboptimal zinc intake in a Western-type diet on the development of vascular inflammation and arterial plaque in apoE knock-out (AEKO) mice. METHODS AND RESULTS: Weanling AEKO and wild-type (WT) controls were given high saturated fat (21% w/w) and high cholesterol (0.15%) semi-synthetic diets containing 3 or 35 mg Zn/kg (AEKO and WT) or 8 mg Zn/kg (AEKO only) for over 6 months. AEKO mice on zinc intakes of 3 and 8 mg Zn/kg (suboptimal zinc) developed significantly (p < 0.05) more aortic plaque than AEKO mice consuming 35 mg Zn/kg (adequate zinc). Circulating levels of interleukin-1ß, interleukin-6 and soluble vascular adhesion molecule-1 were significantly (p < 0.05) raised at the lowest zinc intake in AEKO mice, as compared to zinc-adequate controls. Plasma total cholesterol and total protein were also significantly (p < 0.05) increased at the lowest zinc intake. CONCLUSION: We propose that suboptimal dietary zinc intake raises circulating pro-atherogenic lipoprotein levels that promote vascular inflammation and enhance arterial plaque formation.

Nutritional B vitamin deficiency disrupts lipid metabolism causing accumulation of proatherogenic lipoproteins in the aorta adventitia of ApoE null mice.

SCOPE: Cardiovascular disease is the major cause of death in the world. Low dietary folate, elevated homocysteine, and high circulating cholesterol are risk factors. METHODS AND RESULTS: We investigated whether folate and/or B vitamin deficiency would change lipoprotein and fatty acid metabolism and lipid accumulation in the aorta adventitia of ApoE null mice. Mice (n = 10 per group) were fed a control (C; 4%) or high saturated fat (HF; 21%), and high cholesterol (0.15%) diet for 16 weeks. Folate (F-) or folate, B6 and B12 deficiency (F-B-) were imposed on these diets. Feeding a HF diet increased plasma and liver total cholesterol and HDL cholesterol (two- to threefold; p < 0.05). Total cholesterol increased (twofold; p < 0.05) in aorta adventitial lipid in response to HF. Feeding a diet depleted of folate and B vitamins (F-B-) significantly increased cholesterol accumulation in both liver and aorta adventitial lipid (approximately 50-70%; p < 0.05). Moreover, the proportions of fatty acids in hepatic and adventitial lipid was significantly changed by B vitamin depletion, measured as an increase in saturated fatty acids (approximately 15%) and a decrease (approximately 11%) in monounsaturated fatty acids (p < 0.05). CONCLUSION: B vitamin deficiency perturbs lipid metabolism in ApoE null mice, causing accumulation of proatherogenic cholesterol and fatty acids in the aorta adventitia.

Microanalytical isotope ratio measurements and elemental mapping using laser ablation ICP-MS for tissue thin sections: zinc tracer studies in rats.

The kinetics of zinc absorption, metabolism and excretion is extensively studied by nutritionists. Stable isotopes of zinc can be used to identify body zinc compartments that have different turnover kinetics. Since the compartments might belong to physiological subsections of different organs, there is a need for microsampling analysis to determine isotope ratios of the trace element zinc in tissue samples. Here, we study the feasibility to use laser ablation coupled to quadrupole ICP-MS for the determination of zinc tracers given to rats at different time points with the aim to generate isotope ratio bioimages of heart tissue. A double tracer ((70)Zn and (67)Zn) experiment with rats was designed to label the exchangeable zinc pool as well as the stable zinc pool. The isotope ratios determined by laser ablation ICP-MS were evaluated by additional measurements of tissue digests. Accumulated tracers which made up more than 0.1% of total zinc could be identified in the tissues of the treated rats. It was established that at least 50 measurements from the microsampling were necessary to distinguish between controls and a tracer treated rat resulting in reduced resolution of the bioimage. With the parameters used, features in the tissue thin sections of at least 250 µm(2) in size are necessary to detect the incorporation of a tracer. When different time points have to be measured, higher precisions are required and therefore a larger area needs to be ablated (1 mm(2)). Using the bioimages and pool measurements from one physiological feature, it was possible to show that the aorta cell walls incorporate the zinc tracer at the different time points.

Ginger phytochemicals mitigate the obesogenic effects of a high-fat diet in mice: a proteomic and biomarker network analysis.

SCOPE: Natural dietary anti-obesogenic phytochemicals may help combat the rising global incidence of obesity. We aimed to identify key hepatic pathways targeted by anti-obsogenic ginger phytochemicals fed to mice. METHODS AND RESULTS: Weaning mice were fed a high-fat diet containing 6-gingerol (HFG), zerumbone (HFZ), a characterized rhizome extract of the ginger-related plant Alpinia officinarum Hance (high fat goryankang, HFGK) or no phytochemicals (high-fat control, HFC) for 6 wks and were compared with mice on a low-fat control diet (LFC). Increased adiposity in the HFC group, compared with the LFC group, was significantly (p<0.05) reduced in the HFG and HFGK groups without food intake being affected. Correlation network analysis, including a novel residuals analysis, was utilized to investigate relationships between liver proteomic data, lipid and cholesterol biomarkers and physiological indicators of adiposity. 6-Gingerol significantly increased plasma cholesterol but hepatic farnesyl diphosphate synthetase, which is involved in cholesterol biosynthesis was decreased, possibly by negative feedback. Acetyl-coenzyme A acyltransferase 1 and enoyl CoA hydratase, which participate in the ß-oxidation of fatty acids were significantly (p<0.05) increased by consumption of phytochemical-supplemented diets. CONCLUSION: Dietary ginger phytochemicals target cholesterol metabolism and fatty acid oxidation in mice, with anti-obesogenic but also hypercholesterolemic consequences.

Intervention with fish oil, but not with docosahexaenoic acid, results in lower levels of hepatic soluble epoxide hydrolase with time in apoE knockout mice.

Long-chain n-3 PUFA from fish oil protect against death from CHD but mechanisms are not well understood. Preliminary results indicate that fish oil may affect the enzyme soluble epoxide hydrolase (sEH) and influence inflammatory pathways in a time-dependent manner. In the present study male apoE knockout (Apoe-/-) mice were randomised to three dietary groups receiving a high-fat high-cholesterol diet supplemented with 2 % (w/w) high-oleic acid sunflower-seed (HOSF) oil, DHA oil or fish oil. Livers and proximal aortas were collected on day 2 and on weeks 1, 2, 4 and 10 to determine hepatic sEH levels, hepatic fatty acid composition, hepatic proteome and atherosclerotic plaque size in the aortic root. Intervention with fish oil, but not with DHA, resulted in significantly lower levels of hepatic sEH levels with time compared with HOSF oil. DHA and fish oil caused differential regulation of thirty-five hepatic proteins which were mainly involved in lipoprotein metabolism and oxidative stress. All mice developed atherosclerosis without differences in plaque size between the three groups. Thus EPA may be responsible for lowering levels of hepatic sEH and both fish oil and DHA could beneficially affect lipoprotein metabolism and oxidative stress.

Rapid quantification of aortic lesions in apoE(-/-) mice.

The quantification of aortic lesions is an important end-point analysis for evaluating atherogenesis in mouse models of atherosclerosis. Morphometric methods involving the staining of aorta with a Sudan lysochrome followed by image analysis of the stained lesion area are commonly used. We have developed a more rapid method involving solubilisation of the stain retained by aortic lesions. In 2 separate studies, 5-week-old male apoE(-/-) and C57BL/6 wild-type (apoE(+/+)) mice were given a high fat (21%), Western-type diet for 13, 15 or 25 weeks. At study termination, the descending thoracic aorta (DA) and/or aortic arch (AA) were stained with Oil Red O (ORO). The incorporated stain was extracted using chloroform/methanol (2:1) solvent and quantified by spectrophotometry at 520 nm. In study 1 (13 weeks), ORO stain in the AA and DA of apoE(-/-) mice was 1.9 and 1.4 times higher than background staining of apoE(+/+) aorta tissue, respectively. At 15 and 25 weeks (study 2), ORO stain in the AA of apoE(-/-) mice was 1.9 and 2.5 times higher than the background, respectively. We conclude that the ORO solubilisation technique applied to AA samples is a very useful and rapid method for atherosclerotic lesion quantification.

Aorta protein networks in marginal and acute zinc deficiency.

Human zinc deficiency is a global problem and may influence the development of cardiovascular disease. Our objective was to determine Zn deficiency affected pathways and protein interactions in rat aorta and their likely influence on stress-induced atherogenesis. In two separate studies, rats were given diets acutely (<1 mg Zn/kg) or marginally (6 mg Zn/kg) deficient in Zn. Both studies included Zn adequate controls (35 mg Zn/kg) and the acute deficiency study included a pair-fed group. After 6 wk, proteins from thoracic aorta were separated by 2-DE. Proteins affected by zinc deficiency were identified by principal component analysis. Multiple correlations of identified proteins indicated protein networks of related function. Proteins clusters decreased in zinc deficiency were related to fatty acid and carbohydrate metabolism. Structurally related proteins, including zyxin and over nine transgelin 1 proteins, were either increased or decreased by acute and marginal deficiencies. PKC alpha was significantly decreased in Zn deficiency suggesting that Zn may regulate the phosphorylation of target proteins. Zn deficiency-related changes in structural, carbohydrate and fatty acid-related proteins may be disadvantageous for maintaining vascular health and are consistent with a protective role for zinc in the development of atherosclerosis.

Mononuclear cell metallothionein mRNA levels in human subjects with poor zinc nutrition.

Human zinc deficiency is thought to be prevalent worldwide, particularly in populations with diets low in zinc and animal protein and high in inhibitors of zinc absorption, such as phytic acid. Confirmation of zinc deficiency is, however, difficult in the absence of a reliable and sensitive marker of zinc status. Under controlled conditions, T-lymphocyte metallothionein-2A (MT-2A) mRNA levels change in relation to zinc status and the objective of the present study was to investigate whether these transcript levels could be related to dietary zinc intake, plasma zinc or other biochemical parameters influenced by, or influencing, zinc metabolism in human subjects likely to be zinc deficient. Rural Koreans (n 110, age 50-80 years) with a range of zinc and phytic acid dietary intake were recruited for the study and blood samples were analysed for plasma zinc, HDL, LDL, alpha-tocopherol and thiobarbituric acid reactive substances, mononuclear cell (MNC) MT-2A mRNA, serum protein and albumin, and blood haematocrit, Hb and glucose. Multiple correlation and principal component analysis showed a significant negative correlation between plasma zinc and MNC MT-2A mRNA levels. Female subjects had higher MT-2A transcript levels than males and MT-2A mRNA levels tended to increase with age. There was no significant association between dietary zinc intake or any index of zinc intake relating to dietary inhibitors of zinc absorption. It is concluded that MNC MT-2A mRNA levels cannot be used to predict poor zinc nutrition.

Hepatic responses to dietary stress in zinc- and metallothionein-deficient mice.

Metallothionein (MT) and zinc are both reported to be protective against oxidative and inflammatory stress and may also influence energy metabolism. The role of MT in regulating intracellular labile zinc, thus influencing zinc (Zn)-modulated protein activity, may be a key factor in the response to stress and other metabolic challenges. The objective of this study was to investigate the influence of dietary zinc intake and MT on hepatic responses to a pro-oxidant stress and energy challenge in the form of a high dietary intake of linoleic acid, an omega-6 polyunsaturated fatty acid. Male MT-null (KO) and wild-type (WT) mice, aged 16 weeks, were given semisynthetic diets containing 16% fat and either 5 (marginally zinc-deficient [ZD]) or 35 (zinc-adequate [ZA]) mg Zn/kg. For comparison, separate groups of KO and WT mice were given a rodent chow diet containing 3.36% fat and 86.6 mg Zn/kg. After 4 months on these diets, the body weights of all mice were equal, but liver size, weight, and lipid content were much greater in the animals that consumed semisynthetic diets compared to the chow diet. The increase in liver size was significantly lower in ZA but not ZD KO mice, compared with WT mice. Principally, MT appears to affect the diet-induced increase in liver tissue but it also influences the concentration of hepatic lipid. Plasma levels of C-reactive protein (CRP), a marker of inflammation, were increased by zinc deficiency in WT mice, suggesting that marginal zinc deficiency is proinflammatory. CRP was unaffected by zinc deficiency in KO mice, indicating a role for MT in modulating the influence of zinc. Neither zinc nor MT deficiency affects the level of soluble liver proteins, as determined using two-dimensional (2D) gel proteomics. This study highlights the close association between zinc and MT in the manifestation of stress responses.