John Y. J. Wu

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 as the hepatic and intestinal gene expression of apolipoprotein (apo) A-I was examined in rats and hamsters. Male Sprague-Dawley rats (8 wk old) and Golden Syrian hamsters (7 wk old) were assigned to three dietary treatments: 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 diet for the last 2 days). The dietary treatments lasted for 18 days for rats or 6 wk for hamsters. For the measurement of apoA-I mRNA abundance, hamster apoA-I cDNA was cloned from the small intestine. The full-length 905-base pair cDNA shared ∼80% similarity with the human, rat, and mouse apoA-I cDNAs. Hepatic and plasma Zn levels were reduced in ZD animals but normalized in ZDA rats and increased in ZDA hamsters compared with ZA animals. Zn deficiency reduced plasma apoA-I and 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-I mRNA levels in ZDA rats by 34 and 28%, respectively, higher than ZA rats. Similarly, these levels were decreased by 18 and 25%, respectively, in ZD hamsters but normalized in ZDA hamsters compared with ZA hamsters. In contrast to the alterations of hepatic apoA-I mRNA levels, neither Zn deficiency nor subsequent Zn repletion produced alterations in the intestinal apoA-I mRNA abundance. Data from this study demonstrated that Zn deficiency specifically decreases hepatic apoA-I gene expression, which may at least be partly responsible for the reduction of plasma apoA-I levels.

Apolipoprotein A-I gene expression is regulated by cellular zinc status in Hep G2 cells

The influence of Zn on the expression of the apolipoprotein A-I (apoA-I) gene in Hep G2 cells was examined. Zn depletion was achieved with a low-Zn (ZD) medium prepared from Zn-free growth medium (Opti), a ZD medium containing Chelex 100-extracted fetal bovine serum (CHE), and a medium containing chelator 1,10-phenanthroline (OP). Compared with those for their respective controls, cellular Zn levels were reduced by 55, 48, and 46% and apoA-I mRNA abundances were reduced by 20, 29, and 28% in Opti, CHE, and OP systems, respectively, after one passage in ZD media or 24 h in OP medium. To establish the specificity of Zn treatment, groups of ZD cells were treated with their respective control media for the last 24 h (ZDA) or normal cells were cultured with OP medium supplemented with Zn (OP-Zn). ZDA treatments partially normalized cellular Zn levels in the Opti system and restored or elevated apoA-I mRNA levels in the Opti or CHE system, respectively. Similarly, the OP-Zn treatment restored the cellular Zn and apoA-I mRNA levels. Furthermore, one passage of culture with Zn-supplemented media in both the Opti and CHE systems resulted in higher cellular Zn and apoA-I mRNA levels than those for controls. Most significantly, short-term high-Zn induction to normal cells markedly elevated the cellular Zn (3-fold) and apoA-I mRNA (5-fold) levels. Data derived from this study strongly suggest that the expression of apoA-I is regulated by cellular Zn status.The influence of Zn on the expression of the apolipoprotein A-I (apoA-I) gene in Hep G2 cells was examined. Zn depletion was achieved with a low-Zn (ZD) medium prepared from Zn-free growth medium (Opti), a ZD medium containing Chelex 100-extracted fetal bovine serum (CHE), and a medium containing chelator 1, 10-phenanthroline (OP). Compared with those for their respective controls, cellular Zn levels were reduced by 55, 48, and 46% and apoA-I mRNA abundances were reduced by 20, 29, and 28% in Opti, CHE, and OP systems, respectively, after one passage in ZD media or 24 h in OP medium. To establish the specificity of Zn treatment, groups of ZD cells were treated with their respective control media for the last 24 h (ZDA) or normal cells were cultured with OP medium supplemented with Zn (OP-Zn). ZDA treatments partially normalized cellular Zn levels in the Opti system and restored or elevated apoA-I mRNA levels in the Opti or CHE system, respectively. Similarly, the OP-Zn treatment restored the cellular Zn and apoA-I mRNA levels. Furthermore, one passage of culture with Zn-supplemented media in both the Opti and CHE systems resulted in higher cellular Zn and apoA-I mRNA levels than those for controls. Most significantly, short-term high-Zn induction to normal cells markedly elevated the cellular Zn (3-fold) and apoA-I mRNA (5-fold) levels. Data derived from this study strongly suggest that the expression of apoA-I is regulated by cellular Zn status.

Copper Deficiency Increases Total Protein and Apolipoprotein A-I Synthesis in the Rat Small Intestine

Abstract This study was designed to determine whether an enhanced intestinal synthesis of apolipoprotein (apo) A-I is associated with the hyperapolipoproteinemia observed in copper-deficient rats. Male weanling Sprague-Dawley rats were assigned to two dietary treatments, Cu deficient (0.6 ppm Cu) and Cu adequate (6.0 ppm Cu) for 6 weeks. In vivo studies were then performed after rats were injected with a flooding dose of 150 μM [ 3 H]phenylalanine (PHE, 50 μCi/ml/100 g body wt). Three rats from each treatment were sacrificed at 5, 10, 15, 30, and 60 min postinjection. The small intestine was rapidly rinsed and frozen in liquid N2. In vitro studies were performed by labeling freshly isolated 6-cm segments from duodenum, jejunum, and ileum with [ 3 H]PHE (33 μCi/ml, 49.7 Ci/mmol) in PHE-free minimum essential medium for 7 and 14 min. In vivo and in vitro intestinal samples were sonicated, solubilized in 1% Triton X-100, and centrifuged to provide the detergent soluble fraction for the isolation of nascent apo A-I and total protein. Radioactivities associated with nascent apo A-I isolated by immunoprecipitation and SDS-PAGE, and with total protein precipitated by trichloroacetic acid, were measured to determine the influence of Cu deficiency on nascent apo A-I and total protein synthesis. In the Cu-deficient small intestine, the synthesis of total protein was measured only in the duodenum and was enhanced after 1 hr for the in vivo studies. Moreover, total protein synthesis was enhanced at both 7 and 14 min of the in vitro studies for all three small intestinal segments of the Cu-deficient rats. Apo A-I synthesis was measured only at the jejunum and was also enhanced by Cu deficiency in the in vitro studies. Thus, an increase in intestinal apo A-I synthesis may contribute to the elevated plasma apo A-I level in Cu-deficient rats.

Hepatic translation of apolipoprotein A-I mRNA is apparently enhanced in copper-deficient rats

Male weanling Sprague-Dawley rats were assigned to Cu-deficient (0.6 mg Cu/kg diet) and Cu-adequate (6.0 mg Cu/kg diet) dietary treatments for 6 weeks. Liver post-mitochondrial cytoplasmic extracts were partitioned by centrifugation into polysomes, monosomes, and ribosomal subunits along a linear sucrose gradient. This gradient was further fractionated. The apolipoprotein (apo) A-I mRNA abundance among individual fractions and in the unfractionated cytoplasmic extract were determined by dot blot analysis. The abundance of cytoplasmic apo A-I mRNA was not different between treatments. However, Cu deficiency significantly shifted a portion of cytoplasmic apo A-I mRNA to the more active and larger size polysome fractions. During translation, an increase in the percentage of larger polysomes is indicative of an accelerated rate of initiation. The present data suggest that an enhanced hepatic apo A-I translation efficiency may have contributed to the observed increases in hepatic apo A-I synthesis and plasma apo A-I level in Cu-deficient rats found in previous studies.