Zinc deficiency decreases plasma level and hepatic mRNA abundance of apolipoprotein A-I in rats and hamsters

The influence of Zn deficiency on the plasma level as well asthe hepatic and intestinal gene expression of apolipoprotein (apo) A-Iwas examined in rats and hamsters. Male Sprague-Dawley rats (8 wk old)and Golden Syrian hamsters (7 wk old) were assigned to three dietarytreatments: Zn adequate (ZA, 30 mg Zn/kg diet), Zn deficient (ZD,<0.5 mg Zn/kg diet), and Zn replete (ZDA, ZD animals fed the ZA dietfor the last 2 days). The dietary treatments lasted for 18 days forrats or 6 wk for hamsters. For the measurement of apoA-I mRNAabundance, hamster apoA-I cDNA was cloned from the small intestine. Thefull-length 905-base pair cDNA shared ~80% similarity with thehuman, rat, and mouse apoA-I cDNAs. Hepatic and plasma Zn levels werereduced in ZD animals but normalized in ZDA rats and increased in ZDAhamsters compared with ZA animals. Zn deficiency reduced plasma apoA-Iand hepatic apoA-I mRNA levels 13 and 38%, respectively, in ZD rats.The 2 days of Zn replenishment raised plasma apoA-I and hepatic apoA-ImRNA levels in ZDA rats by 34 and 28%, respectively, higher than ZArats. Similarly, these levels were decreased by 18 and 25%,respectively, in ZD hamsters but normalized in ZDA hamsters comparedwith ZA hamsters. In contrast to the alterations of hepatic apoA-I mRNAlevels, neither Zn deficiency nor subsequent Zn repletion producedalterations in the intestinal apoA-I mRNA abundance. Data from thisstudy demonstrated that Zn deficiency specifically decreases hepaticapoA-I gene expression, which may at least be partly responsible forthe reduction of plasma apoA-I levels.

     

Zinc status affects p53, gadd45, and c-fos expression and caspase-3 activity in human bronchial epithelial cells

This study was designed to examine theinfluence of zinc depletion and supplementation on the expression ofp53 gene, target genes of p53, andcaspase-3 activity in normal human bronchial epithelial (NHBE) cells. Aserum-free, low-zinc medium containing 0.4 µmol/l of zinc [zincdeficient (ZD)] was used to deplete cellular zinc over one passage. Inaddition, cells were cultured for one passage in media containing 4.0 µmol/l of zinc [zinc normal (ZN)], which represents normal cultureconcentrations (Clonetics); 16 µmol/l of zinc [zinc adequate (ZA)],which represents normal human plasma zinc levels; or 32 µmol/l ofzinc [zinc supplemented (ZS)], which represents the high end ofplasma zinc levels attainable by oral supplementation in humans.Compared with ZN cells, cellular zinc levels were 76% lower in ZDcells but 3.5-fold and 6-fold higher in ZA and ZS cells, respectively.Abundances of p53 mRNA and nuclear p53 protein were elevatedin treatment groups compared with controls (ZN). For p53mRNA abundance, the highest increase (3-fold) was observed in ZD cells.In contrast, the highest increase (17-fold) in p53 nuclearprotein levels was detected in ZS cells. Moreover, gadd45mRNA abundance was moderately elevated in ZD and ZA cells and was notaltered in ZS cells compared with ZN cells. Furthermore, the onlyalteration in c-fos mRNA and caspase-3 activity was thetwofold increase and the 25% reduction, respectively, detected in ZScompared with ZN cells. Thus p53, gadd45, andc-fos and caspase-3 activity appeared to be modulated bycellular zinc status in NHBE cells.

     

Effects of zinc deficiency and zinc supplementation on homocysteine levels and related enzyme expression in rats

Methionine synthase (MS) and betaine-homocysteine methyltransferase (BHMT) are both zinc (Zn)-dependent methyltransferases and involved in the methylation of homocysteine. The objective of this study was to investigate the effects of dietary Zn supply on homocysteine levels and expression of the two enzymes in growing rats. Male weanling Sprague-Dawley rats were assigned randomly to four dietary groups (n = 8/group) for 3 weeks: Zn deficient (ZD; <1 mg Zn/kg); Zn control (ZC; 30 mg Zn/kg); Zn supplemented (ZS; 300 mg Zn/kg); pair fed (PF; 30 mg Zn/kg) to the ZD group. Serum and femur Zn concentrations were 83% and 58% lower in ZD, and 49% and 62% higher in ZS compared to ZC (P < 0.001), respectively. The ZD rats had lower feed intake (37%), body weight gains (45%), liver (43%) and kidney (31%) weights than those of ZC (P < 0.001), but these parameters in ZD were not significantly different from the PF controls. Serum homocysteine concentrations were 65% higher in ZD compared to PF (P < 0.05), and there was no significant difference in serum folate levels between ZD and PF groups. The mRNA expression of liver and kidney MS was 57% and 38% lower in ZD than PF (P < 0.001), respectively. Hepatic and renal BHMT mRNA levels were not altered in ZD compared to controls. The aforementioned measurements were not significantly different between ZS and ZC groups, except Zn levels. These results demonstrated that homocysteine homeostasis appeared to be disturbed by Zn deficiency but not Zn supplementation, and elevated serum homocysteine might be due to reduced expression of MS during Zn deficiency.     

Effects of Zinc Supplementation and Deficiency on Bone Metabolism and Related Gene Expression in Rat

One hundred male rats were randomly divided into four groups (n = 25) and fed a Zn-adequate diet (ZA, 46.39 mg/kg), Zn-deficient diet (ZD, 3.20 mg/kg), Zn-overdose diet (ZO, 234.39 mg/kg), or were pair-fed a Zn-adequate diet (PF) for 5 weeks, respectively. The body weight, femur weight, and activity of alkaline phosphatase (ALP) were reduced in the ZD group but were increased in the ZO group. Zn concentrations in both liver and femur were elevated in the ZO group, whereas femur Zn was decreased in the ZD group. The concentrations of calcium and phosphorus were lower in the ZD than those in other groups. Serum calcium concentration was decreased in the ZD. The relative expression level of ALP was decreased in both ZD and PF, and no significant differences were observed between ZO and ZA. Insulin-like growth factor-I (IGF-I) mRNA level was reduced in the ZD but unchanged in the ZO and PF group. Zn deficiency also decreased ALP mRNA level as compared with that of PF group. Carbonic anhydrase II mRNA level was not affected by Zn. Nevertheless, dietary Zn influenced the growth, bone metabolism, and expression of IGF-I and ALP in male growing rats.     

p53 protein and p21 mRNA levels and caspase-3 activity are altered by zinc status in aortic endothelial cells

The influence ofzinc status on the levels of p53, as well as downstream targetsof p53 in cell repair and survival, was examined in human aorticendothelial cells (HAECs). A serum-reduced low-zinc medium (ZD) wasused to deplete zinc over one passage. Other treatments includedzinc-normal control (ZN), zinc-adequate (ZA), and zinc-supplemented (ZS) treatment with 3.0, 16.0, and 32.0 µM zinc, respectively. Cellular zinc levels in the ZD cells were 64% of ZN controls; levelsin the ZA cells were not different, but levels in ZS cells weresignificantly higher (40%) than in ZN cells. No difference in p53 mRNAabundance was detected among all treatments; however, p53 nuclearprotein levels were >100% higher in the ZD and ZS cells and almost200% higher in the ZA cells than in ZN controls. In addition, p21 mRNAabundance, a downstream target of p53 protein, was increased in the ZScells compared with both the ZN control and ZD cells. In the ZS cells,bax and mcl-1 were also ~50% higher compared with ZN controls,whereas bcl-2 mRNA was increased compared with ZA cells. Moreover,caspase-3 activity of ZD cells was not different from that of ZNcontrols but was reduced to 83 and 69% of ZN controls in ZA and ZScells, respectively. Thus p53 protein and p53 downstream target genesappeared to be modulated by intracellular zinc status in HAECs.

     

Angiotensin II type-1 receptor antagonist attenuates LPS-induced acute lung injury

Angiotensin II is able to trigger inflammatory responses through an angiotensin II type 1 (AT1) receptor. The role of AT1 receptor in acute lung injury (ALI) is poorly understood. Mice were randomly divided into three groups (n = 40 each groups): NS group; LPS group (2 mg/kg LPS intratracheally); and LPS + ZD 7155 group, 10 mg/kg ZD 7155 (an AT1 receptor antagonist) intraperitoneally 30 min prior to LPS exposure. Samples from the lung were isolated and assayed for histopathology analyses or proinflammatory gene expressions, angiotensin II receptors expressions and nuclear factors activities. LPS exposure resulted in severe ALI, elevated levels of TNF-α and IL-1β mRNA expressions, and increased activities of NF-κB and activated protein (AP)-1. Upregulation of AT1 receptor and down-regulation of AT2 receptor were also observed after LPS challenge. Pretreatment with ZD 7155 significantly inhibited the increase of AT1 receptor expression and upregulated AT2 receptor expression. ZD 7155 also reduced the mRNA expression of TNF-α and IL-1β, inhibited the activation of NF-κB and AP-1, and improved lung histopathology. These findings suggest that antagonism of AT1 receptor inhibits the activation of NF-κB and AP-1 in the lung, which may mediate the release of TNF-α and IL-1β and contribute to LPS-induced ALI.     

Structure, evolution, and regulation of chicken apolipoprotein A-I

A full-length cDNA clone for the precursor form of chicken liver apolipoprotein A-I (apoA-I) was isolated by antibody screening of a chicken liver cDNA library in the expression vector lambda gt11. The complete nucleotide sequence and predicted amino acid sequence of this clone is presented. The identity of the clone was confirmed by comparison with partial amino acid sequences for chicken apolipoprotein A-I. Chicken preproapolipoprotein A-1 consists of an 18-amino acid prepeptide, a 6-amino acid propeptide, and 240 amino acids of mature protein. The sequence of the protein is homologous to mammalian apoA-I and is highly internally repetitive, consisting largely of 11-amino acid repeats predicted to have an amphipathic alpha-helical structure. The sequence of the propeptide (Arg-Ser-Phe-Trp-Gln-His) differs in two positions from that of mammalian apoA-I. The mRNA for chicken apoA-I is about 1 kilobase in length and is expressed in a variety of tissues including liver, intestine, brain, adrenals, kidneys, heart, and muscle. This quantitative tissue distribution has been determined and is similar to that observed for mammalian apoE and different from that of mammalian apoA-I mRNA. This reinforces the concept that avian apoA-I performs functions analogous to those of mammalian apoE. Moreover, comparisons revealed sequences of chicken apoA-I similar to the region of mammalian apoE responsible for interaction with cellular receptors. Previous studies have demonstrated striking changes in the rates of synthesis of apoA-I in breast muscle during development and in optic nerve after retinal ablation. We now demonstrate that these changes are paralleled by changes in mRNA levels. ApoA-I mRNA levels increase approximately 50-fold in breast muscle between 14 days postconception and hatching and then decrease about 15-fold to adult levels. The levels of apoA-I mRNA increase about 3-fold in optic nerve following retinal ablation. ApoA-I mRNA is also found in the brain in the absence of nerve injury. This may indicate that locally synthesized apoA-I has a routine or housekeeping function in lipid metabolism in the central nervous system.     

Redistribution of tissue zinc pools during lactation and dyshomeostasis during marginal zinc deficiency in mice

Zinc (Zn) requirements are increased during lactation. Increased demand is partially met through increased Zn absorption from the diet. It is estimated that 60–80% of women of reproductive age are at risk for Zn deficiency due to low intake of bioavailable Zn and increased demands during pregnancy and lactation. How Zn is redistributed within the body to meet the demands of lactation, and how Zn deficiency affects this process, is not understood. Female C57bl/6J mice were fed a control (ZA; 30 mg Zn/kg) or a marginally Zn deficient (ZD; 15 mg Zn/kg) diet for 30 days prior to mating through mid-lactation and compared with nulliparous mice fed the same diets. While stomach and plasma Zn concentration increased during lactation in mice fed ZA, mice fed ZD had lower stomach Zn concentration and abrogated plasma Zn levels during lactation. Additionally, femur Zn decreased during lactation in mice fed ZA, while mice fed ZD did not experience this decrease. Furthermore, red blood cell, pancreas, muscle and mammary gland Zn concentration increased, and liver and adrenal gland Zn decreased during lactation, independent of diet, while kidney Zn concentration increased only in mice fed ZD. Finally, maternal Zn deficiency significantly increased the liver Zn concentration in offspring but decreased weight gain and survival. This study provides novel insight into how Zn is redistributed to meet the increased metabolic demands of lactation and how marginal Zn deficiency interferes with these homeostatic adjustments.     

Angiotensin II type-1 receptor antagonist attenuates LPS-induced acute lung injury

Angiotensin II is able to trigger inflammatory responses through an angiotensin II type 1 (AT1) receptor. The role of AT1 receptor in acute lung injury (ALI) is poorly understood. Mice were randomly divided into three groups (n = 40 each groups): NS group; LPS group (2 mg/kg LPS intratracheally); and LPS + ZD 7155 group, 10 mg/kg ZD 7155 (an AT1 receptor antagonist) intraperitoneally 30 min prior to LPS exposure. Samples from the lung were isolated and assayed for histopathology analyses or proinflammatory gene expressions, angiotensin II receptors expressions and nuclear factors activities. LPS exposure resulted in severe ALI, elevated levels of TNF-α and IL-1β mRNA expressions, and increased activities of NF-κB and activated protein (AP)-1. Upregulation of AT1 receptor and down-regulation of AT2 receptor were also observed after LPS challenge. Pretreatment with ZD 7155 significantly inhibited the increase of AT1 receptor expression and upregulated AT2 receptor expression. ZD 7155 also reduced the mRNA expression of TNF-α and IL-1β, inhibited the activation of NF-κB and AP-1, and improved lung histopathology. These findings suggest that antagonism of AT1 receptor inhibits the activation of NF-κB and AP-1 in the lung, which may mediate the release of TNF-α and IL-1β and contribute to LPS-induced ALI.     

Cytoprotective mechanisms in rats lung parenchyma with zinc deprivation

Suboptimal intake of Zinc (Zn) is one of the most common worldwide nutritional problems. The aim of this study is to provide new evidence on the relation between moderate Zn restriction, and cytoprotective functions in airway epithelium. We analyzed the effect of moderate Zn deficiency (ZD) on the expression of several pro and anti-apoptotic proteins and cytoprotective factors (Hsp27 and Hsp 70i), as well as the effect of restoring Zn during the refeeding period. Adult male rats were divided into three groups: Zn-adequate control group, Zn-deficient group and Zn-refed group. Our previous findings showed an important oxidative and nitrosative stress during ZD, this situation is accompanied by inflammation and alterations in the expression of matrix extracellular proteins. We observed a strong immunopositive area of anti and pro-apoptotics proteins in ZD groups. The mRNA levels of Nrf-2, Bax and Bad were increased in ZD, while in ZD refed group its levels were similar to the control values. The increased expression of Nrf-2 is likely to be critical for protection of lung under inflammatory process triggered during ZD. Hsp27 and Hsp 70i showed an increase of immunostaining area but they were not significant. During the supplementation period, heat-shock proteins increased significantly. In conclusion, our results provide further evidence of the pathways involved in cytoprotection and apoptosis caused by ZD. Additional studies are required in order to investigate whether Hsp27 and Hsp70 are consistently associated with cellular stress and inflammation in lung. There may be a beneficial role for improved Zn nutrition or Zn supplements early in lung pathology.     

Factors influencing somatic embryo maturation, high frequency germination and plantlet formation in Terminalia chebula Retz.

The factors influencing somatic embryo maturation, high frequency somatic embryo germination, and plantlet formation were studied in Terminalia chebula Retz. Maturation of somatic embryo were influenced by a number of factors such as in vitro culture passage, concentrations of sucrose, levels of abscisic acid (ABA), basal media and media additive combinations. Maximum frequency of somatic embryo maturation (57.22 ± 2.02), was obtained on MS medium supplemented with 50 g/l sucrose. Different factors such as strengths of MS nutrients, plant growth regulators, media additives and their combinations controlling somatic embryo germination and plantlet formation were studied. High frequency of germination and plantlet formation (58.80 ± 1.47) were achieved by subsequent subculture of mature somatic embryos on MS medium containing 30 g/l sucrose and 0.5 mg/l benzyladenine (BA). However, although duration of in vitro passage of the callus tissue was critical, contribution of the combinations of plant growth regulators and media additives showed nugatory effect on somatic embryo maturation and germination as evident from variable responses.     

Apolipoprotein E gene expression is reduced in apolipoprotein A-I transgenic mice

The levels of plasma apolipoprotein (apo) E, an anti-atherogenic protein involved in mammalian cholesterol transport, were found to be 2-3 fold lower in mice over-expressing human apoA-I gene. ApoE is mainly associated with VLDL and HDL-size particles, but in mice the majority of the apoE is associated with the HDL particles. Over-expression of the human apoA-I in mice increases the levels of human apoA-I-rich HDL particles by displacing mouse apoA-I from HDL. This results in lowering of plasma levels of mouse apoA-I. Since plasma levels of apoE also decreased in the apoA-I transgenic mice, the mechanism of apoE lowering was investigated. Although plasma levels of apoE decreased by 2-3 fold, apoB levels remained unchanged. As expected, the plasma levels of human apoA-I were almost 5-fold higher in the apoAI-Tg mice compared to mouse apoA-I in WT mice. If the over-expression of human apoA-I caused displacement of apoE from the HDL, the levels of hepatic apoE mRNA should remain the same in WT and the apoAI-Tg mice. However, the measurements of apoE mRNA in the liver showed 3-fold decreases of apoE mRNA in apoAI-Tg mice as compared to WT mice, suggesting that the decreased apoE mRNA expression, but not the displacement of the apoE from HDL, resulted in the lowering of plasma apoE in apoAI-Tg mice. As expected, the levels of hepatic apoA-I mRNA (transgene) were 5-fold higher in the apoAI-Tg mice. ApoE synthesis measured in hepatocytes also showed lower synthesis of apoE in the apoAI-Tg mice. These studies suggest that the integration of human apoA-I transgene in mouse genome occurred at a site that affected apoE gene expression. Identification of this locus may provide further understanding of the apoE gene expression.     

Extracellular nucleotides inhibit insulin receptor signaling, stimulate autophagy and control lipoprotein secretion

Hyperglycemia is associated with abnormal plasma lipoprotein metabolism and with an elevation in circulating nucleotide levels. We evaluated how extracellular nucleotides may act to perturb hepatic lipoprotein secretion. Adenosine diphosphate (ADP) (>10 μM) acts like a proteasomal inhibitor to stimulate apoB100 secretion and inhibit apoA-I secretion from human liver cells at 4 h and 24 h. ADP blocks apoA-I secretion by stimulating autophagy. The nucleotide increases cellular levels of the autophagosome marker, LC3-II, and increases co-localization of LC3 with apoA-I in punctate autophagosomes. ADP affects autophagy and apoA-I secretion through P2Y(13). Overexpression of P2Y(13) increases cellular LC3-II levels by ~50% and blocks induction of apoA-I secretion. Conversely, a siRNA-induced reduction in P2Y(13) protein expression of 50% causes a similar reduction in cellular LC3-II levels and a 3-fold stimulation in apoA-I secretion. P2Y(13) gene silencing blocks the effects of ADP on autophagy and apoA-I secretion. A reduction in P2Y(13) expression suppresses ERK1/2 phosphorylation, increases the phosphorylation of IR-β and protein kinase B (Akt) >3-fold, and blocks the inhibition of Akt phosphorylation by TNFα and ADP. Conversely, increasing P2Y(13) expression significantly inhibits insulin-induced phosphorylation of insulin receptor (IR-β) and Akt, similar to that observed after treatment with ADP. Nucleotides therefore act through P2Y(13), ERK1/2 and insulin receptor signaling to stimulate autophagy and affect hepatic lipoprotein secretion.