CuD Rats Research Articles

AIN‐76 and AIN‐93 diets do not provide adequate iron for perinatal development

Iron supplementation during pregnancy and neonatal development remains controversial. Rodent models intending to evaluate trace element deficiencies and interactions should rely on an optimal control diet. When the AIN‐93 formulation was developed it dealt primarily with changes in carbohydrate, lipid, and calcium levels; iron recommendations remained the same as its predecessor diet, AIN‐76A, at 35 mg/kg. Increasing the dietary iron to 75 mg/kg (a level recommended by NRC guidelines) to pregnant Sprague Dawley rats, or postweanling males demonstrated that iron status is improved in dams, pups, as well as rapidly growing males. This is based on measurements of hepatic iron, plasma iron, and hemoglobin concentration. Phenotype of copper‐deficient (CuD) dams and pups fed modified AIN‐76A diets were different than CuD rats fed modified AIN‐93G diets with the higher iron level compared to copper‐adequate (CuA) controls. Hepatic iron was no longer augmented in the CuD groups. Additionally plasma and brain iron differences were attenuated. For example, brain iron deficiency evaluated by augmented transferrin receptor expression in CuD rats was not observed in the CuD pups reared by CuD dams fed the modified AIN‐93G diets with extra iron. It may be prudent to reevaluate the composition of standard “control” diets used in rodent models.Supported by International Copper Association.

Biomarker erythrocyte copper chaperone for superoxide dismutase (CCS) is higher following marginal copper deficiency

There is a critical need to identify an accurate copper biomarker to diagnose and treat adult onset copper deficiency. Prevalence is rising due to secondary impacts of antagonists such as zinc and popularity of bariatric surgery. Marginal copper deficiency (CuM) and iron deficiency (FeD) were studied in Sprague Dawley rat and Swiss Webster mouse models using modified AIN‐93G diets. Red blood cell (RBC) superoxide dismutase (Sod1) and plasma ceruloplasmin (Cp) protein were found to be altered by both FeD and copper deficiency (CuD), while CCS and CCS/Sod1 ratio were found only to be altered in CuD rats; and importantly, after only 1 week of treatment in post weanling males. RBC CCS levels were highly negatively correlated, r = −0.91, with liver copper in CuD, CuM and copper adequate (CuA) rats. Post weanling CuD male mice reared in plastic cages for 5 weeks had no significant reduction in liver copper, plasma Cp protein, or RBC Sod1 compared to CuA mice but CuD mice displayed 2‐fold higher RBC CCS. CuD adult male mice with no significant changes in Cp activity and protein after 5 weeks of dietary treatment displayed augmented RBC CCS. Additional research is needed to establish that erythrocyte CCS is a suitable copper biomarker for humans but RBC CCS appears to be a robust marker for marginal copper deficiency in rodent models.Supported by the International Copper Association and USDA NIFA 2006‐35200‐17378.

Rapid alteration in rat red blood cell copper chaperone for superoxide dismutase after marginal copper deficiency and repletion

There is increased incidence of human copper deficiency (CuD). A sensitive and reliable blood biomarker may reveal additional cases of marginal deficiency. Two experiments were designed to test the hypothesis that the copper chaperone for superoxide dismutase (CCS) would be a robust marker after marginal CuD. Experiment 1 used weanling male Sprague-Dawley rats that were offered a CuD diet for 4 weeks, and samples were evaluated after 1, 2, and 4 weeks and compared with copper-adequate (CuA) controls. Furthermore, iron-deficient rats were included for comparison after 2 weeks of depletion. Red blood cell and plasma cuproenzymes were evaluated through Western blot analysis. Superoxide dismutase (Sod1) and ceruloplasmin protein were found to be altered by both iron and CuD, whereas CCS and CCS/Sod1 ratio were found to only be altered only in CuD rats and, importantly, after only 1 week of treatment. Two weeks on CuA diet restored cuproenzyme levels to control values after 4 weeks of CuD depletion. In experiment 2, marginal CuD (CuM) rats were compared with CuA and CuD rats after 2 weeks of treatment. Superoxide dismutase, ceruloplasmin, and CCS/Sod1 abundances were lower in CuM and CuD groups compared with CuA rats, but there was no statistical difference between CuM and CuD rats. However, CCS was statistically different between all groups, and abundance highly correlated with liver copper concentration. Results suggest that red blood cell CCS may be an excellent biomarker for diagnosis of rapid and marginal CuD.

Splenic iron overload does not correlate with reduced expression of GPI‐ceruloplasmin following dietary copper deficiency

Many believe anemia following copper deficiency is due to impairment in iron efflux from intestine and storage sites in liver and spleen because copper is necessary for ferroxidase activity of multicopper oxidases hephaestin and ceruloplasmin (sCp). Prior work in brain discovered a splice variant of Cp anchored by glycosylphosphatidylinositol (GPI-Cp). Studies were conducted in Sprague Dawley rats to investigate GPI-Cp. Robust mRNA levels of GPI-Cp were detected in multiple rodent tissues in addition to brain. Purified membrane preparations detected immunoreactive Cp protein, especially enriched in spleen and kidney. Plasma from anemic dietary copper-deficient (CuD) rats contained no detectable sCp oxidase activity and reductions (60–90%) of sCp protein. Purified spleen membranes of CuD rats contained marked (>50%) reductions in GPI-Cp abundance. Surprisingly, however, lower non-heme iron concentrations compared to copper-adequate controls were detected. Perhaps the augmentation in ferroportin, the iron exporter, in CuD rats compensates for lower levels of the multicopper oxidase GPI-Cp. Livers of CuD rats also contained lower GPI-Cp levels but no iron augmentation. Additional research is needed to determine the mechanism for copper deficiency induced anemia but reduced sCp and GPI-Cp do not appear to be main factors in this pathology. Supported by NIH HD-39708 and USDA NIFA 2006-35200-17378.

Glycosylphosphatidylinositol-linked ceruloplasmin is expressed in multiple rodent organs and is lower following dietary copper deficiency

Ceruloplasmin (Cp), a multicopper ferroxidase, is expressed as both a secreted (sCp) plasma enzyme from the liver and a membrane-bound glycosylphosphatidylinositol-anchored (GPI-Cp) splice variant protein. Cp is thought to be essential for iron mobilization as selective iron overload occurs in aceruloplasminemia in humans and in Cp null mice. Dietary copper-deficient (CuD) rodents have near total loss of Cp activity, severe loss of Cp protein and develop anemia. Hepatic iron augmentation is often observed, suggesting that loss of Cp function may be correlated with anemia. The impact of CuD treatment on GPI-Cp has not previously been evaluated. Our hypothesis was that CuD rodents would have lower levels of GPI-Cp and this would correlate with higher tissue iron retention. In these studies, GPI-Cp was detected in purified membranes of multiple organs of rats and mice but not Cp -/- mice. Immunoreactive Cp protein was released with phosphatidylinositol phospholipase C treatment and expressed ferroxidase activity. Following perinatal and postnatal copper restriction, GPI-Cp was markedly lower in the spleen and modestly lower in the liver of CuD rats and mice, when compared with copper-adequate (CuA) rodents. However, spleen non-heme iron (NHI) was lower in CuD than CuA rats, and not different in CuD mice. Hepatic iron was higher only in CuD mice. Spleen and liver membranes of CuD rats expressed augmented levels of ferroportin, the iron efflux transporter, which may explain lower NHI content in the spleen of CuD rats despite a greater than 50% lower level of the multicopper ferroxidase GPI-Cp. Spleen and liver levels of GPI-Cp mRNA were not impacted in CuD rats, suggesting that turnover rather than biosynthesis may explain the lower steady-state levels of GPI-Cp following dietary copper restriction. Lower GPI-Cp did not correlate with tissue iron retention and thus the role, if any, of Cp in anemia of copper deficiency is unknown.

Anemic Copper-Deficient Rats, but Not Mice, Display Low Hepcidin Expression and High Ferroportin Levels1–3

The transmembrane protein ferroportin (Fpn) is essential for iron efflux from the liver, spleen, and duodenum. Fpn is regulated predominantly by the circulating iron regulatory hormone hepcidin, which binds to cell surface Fpn, initiating its degradation. Accordingly, when hepcidin concentrations decrease, Fpn levels increase. A previous study found that Fpn levels were not elevated in copper-deficient (CuD) mice that had anemia, a condition normally associated with dramatic reductions in hepcidin. Lack of change in Fpn levels may be because CuD mice do not display reduced concentrations of plasma iron (holotransferrin), a modulator of hepcidin expression. Here, we examined Fpn protein levels and hepcidin expression in CuD rats, which exhibit reduced plasma iron concentrations along with anemia. We also examined hepcidin expression in anemic CuD mice with normal plasma iron levels. We found that CuD rats had higher liver and spleen Fpn levels and markedly lower hepatic hepcidin mRNA expression than did copper-adequate (CuA) rats. In contrast, hepcidin levels did not differ between CuD and CuA mice. To examine potential mediators of the reduced hepcidin expression in CuD rats, we measured levels of hepatic transferrin receptor 2 (TfR2), a putative iron sensor that links holotransferrin to hepcidin production, and transcript abundance of bone morphogenic protein 6 (BMP6), a key endogenous positive regulator of hepcidin production. Diminished hepcidin expression in CuD rats was associated with lower levels of TfR2, but not BMP6. Our data suggest that holotransferrin and TfR2, rather than anemia or BMP6, are signals for hepcidin synthesis during copper deficiency.

Levels of plasma ceruloplasmin protein are markedly lower following dietary copper deficiency in rodents

Ceruloplasmin (Cp) is a multicopper oxidase and the most abundant copper binding protein in vertebrate plasma. Loss of function mutations in humans or experimental deletion in mice result in iron overload consistent with a putative ferroxidase function. Prior work suggested plasma may contain multiple ferroxidases. Studies were conducted in Holtzman rats ( Rattus norvegicus), albino mice ( Mus musculus), Cp−/− mice, and adult humans ( Homo sapiens) to investigate the copper–iron interaction. Dietary copper-deficient (CuD) rats and mice were produced using a modified AIN-76A diet. Results confirmed that o-dianisidine is a better substrate than paraphenylene diamine (PPD) for assessing diamine oxidase activity of Cp. Plasma from CuD rat dams and pups, and CuD and Cp−/− mice contained no detectable Cp diamine oxidase activity. Importantly, no ferroxidase activity was detectable for CuD rats, mice, or Cp−/− mice compared to robust activity for copper-adequate (CuA) rodent controls using western membrane assay. Immunoblot protocols detected major reductions (60–90%) in Cp protein in plasma of CuD rodents but no alteration in liver mRNA levels by qRT-PCR. Data are consistent with apo-Cp being less stable than holo-Cp. Further research is needed to explain normal plasma iron in CuD mice. Reduction in Cp is a sensitive biomarker for copper deficiency.

Perinatal Copper Deficiency Alters Rat Cerebellar Purkinje Cell Size and Distribution

Copper is required for activity of several key enzymes and for optimal mammalian development, especially within the central nervous system. Copper-deficient (CuD) animals are visibly ataxic, and previous studies in rats have demonstrated impaired motor function through behavioral experiments consistent with altered cerebellar development. Perinatal copper deficiency was produced in Holtzman rat dams by restricting dietary copper during the last two thirds of gestation and lactation. Male offspring were evaluated at postnatal day 25. Compared to cerebella from copper-adequate pups, the CuD pups had larger Purkinje cell (PC) size and irregularities in the Purkinje cell monolayer. These results suggest that the ataxic behavioral phenotype of CuD rats may result from disrupted inhibitory pathways in the cerebellum. A similar PC phenotype is seen in Menkes disease and in mottled mouse mutants with genetic copper deficiency, suggesting that copper deficiency and not just specific loss of ATP7A function is responsible.

Oxidative stress in cardiac mitochondria caused by copper deficiency may be insufficient to damage mitochondrial proteins

Copper (Cu) deficiency may promote the generation of reactive oxygen species (ROS) by the mitochondrial electron transport chain through inhibition of cytochrome c oxidase (CCO) and increased reduction of respiratory complexes upstream from CCO. In the present study, respiration, H2O2 production and aconitase activity were measured in isolated heart mitochondria from weanling rats fed diets that were either deficient (CuD, <1ppm Cu), marginally deficient (CuM, 1.5 ppm Cu) or adequate (CuA, 6 ppm Cu) in Cu for 8 weeks. State 4 respiration (μmole O2 ·min−1 ·mg−1) in CuD rats (2.81±0.71) was lower (P<0.05) than in CuM (3.74±0.88) or CuA rats (3.72±0.78). H2O2 production (nmol · min−1 ·mg−1) in the presence of rotenone was marginally higher (p=0.07) in CuD rats (11.2±4.1) than in CuM rats (8.5±2.4) or CuA rats (8.2±2.6). However, aconitase activity (mU ·mg−1) was higher (P<0.05) in CuD rats (58.5±12.5) than in CuM (25.8±7.0) or CuA rats (28.4±9.3). Aconitase is readily inactivated by ROS and these findings suggest that even though severe Cu deficiency impairs mitochondrial function, mitochondrial oxidative stress is not elevated to a level that leads to oxidative inactivation of susceptible mitochondrial enzymes. Enhancement of aconitase activity may reflect enhanced TCA cycle activity for maintenance of cardiac energy production during Cu deficiency.Supported by USDA‐ARS

Copper deficiency increases ferroportin expression in rats

Ferroportin is a transmembrane iron‐export protein that plays an important role in dietary iron absorption and iron recycling from senescent red blood cells. Ferroportin expression increases with tissue iron loading but decreases in response to hepcidin, the circulating iron‐regulatory hormone that binds to ferroportin, causing its internalization and degradation. Hepcidin expression increases with iron loading but decreases during anemia. We examined ferroportin and hepcidin expression in copper‐deficient (CuD) rats, which have elevated liver iron levels but are anemic. We compared CuD and copper‐adequate (CuA) animals (n=4/group) after one month of treatment postweaning. Hemoglobin levels were lower in CuD than in CuA rats (101 vs. 159 g/L). Western blot analysis revealed that ferroportin levels increased in CuD rats relative to CuA rats by 4‐ and 1.6‐fold in liver and spleen, respectively (P<0.02). Increased ferroportin expression in CuD rat tissues was associated with increased ferroportin transcript abundance, as measured by qRT‐PCR. Copy number of hepatic hepcidin mRNA was more than 50‐fold lower in CuD than CuA rats despite a doubling of liver iron. We conclude that tissue ferroportin levels increase in CuD rats because of diminished hepcidin levels and increased ferroportin mRNA abundance.

Signs of iron deficiency in copper‐deficient and control rats fed bovine hemoglobin as the sole source of iron

We determined whether hemoglobin (Hgb) as the sole source of dietary Fe could sustain normal Fe status in growing rats. Because adequate Cu status is required for efficient Fe absorption in the rat, we also determined the effects of Cu deficiency on Fe status of rats fed Fe as Hgb. One group of 24 rats was fed a diet containing 5.0 mg Cu/kg (CuA) and another 24 were fed a diet containing <0.5 mg Cu/kg (CuD). Eight rats in each Cu group were fed 35 mg Fe/kg as FeSO4; another group was fed 35 mg Fe/kg as bovine Hgb; and a third group was fed 70 mg Fe/kg as Hgb. After 4 wk, CuD rats were anemic compared with CuA rats. Rats fed Hgb at 35 mg Fe/kg were anemic, but those fed Hgb at 70 mg Fe/kg were not. Regardless of the Fe source, Cu was lower (P<0.001) in the mucosa, serum, liver, spleen, and kidney of CuD rats when compared with that in CuA rats. Mucosal Fe and liver Fe were significantly (P<0.001) elevated, but serum (P<0.001) and kidney (P<0.040) Fe were lower in CuD rats, compared with CuA rats. Cu deficiency had no effect on spleen Fe. Feeding Fe as Hgb at 35 mg/kg lowered (P<0.001) Fe in serum and spleen compared with feeding Fe as FeSO4; however, these parameters were normal in rats fed Hgb at 70 mg Fe/kg. Regardless of the dietary Fe level as Hgb, mucosal and liver Fe were significantly lower than that in rats fed FeSO4. This study showed that adequate Fe status in the growing rat could not be maintained with Hgb as the sole source of Fe.

Repletion of Copper-Deficient Rats with Dietary Copper Restores Duodenal Hephaestin Protein and Iron Absorption

Copper (Cu) deficiency in rats reduces the relative concentration of duodenal hephaestin (Hp), reduces iron (Fe) absorption, and causes anemia. An experiment was conducted to determine whether these effects could be reversed by dietary Cu repletion. Five groups of eight weanling male rats each were used. Group 1 was fed a Cu-adequate diet (5.0 mg Cu/kg; CuA) and Group 2 was fed a Cu-deficient diet (0.25 mg Cu/kg; CuD) for 28 days. The rats were fed 1.0 g each of their respective diets labeled with 59Fe (37 kBq/g), and the amount of label retained was measured one week later by whole-body-counting (WBC). Group 3 was fed a CuA diet and Groups 4 and 5 were fed a CuD diet for 28 days. Group 5 was then fed the CuA diet for another week while Groups 3 and 4 continued on their previous regimens. Rats in Groups 3, 4, and 5 were fed 1.0 g of diet labeled with 59Fe, and the amount of label retained was measured by WBC one week later. Rats were killed and duodenal enterocytes isolated for Hp protein analysis, whole blood was analyzed for hematological parameters, and various organs for 59Fe content. CuD rats absorbed less (P<0.05) Fe than CuA rats, the relative amount of duodenal Hp was less (P<0.05) in CuD rats, and the CuD rats developed anemia. After the CuD rats had been repleted with Cu for one week, Fe retention rose to values even higher (P<0.05) than those in CuA rats. After two weeks, the relative amount of duodenal Hp was higher (P<0.05) than normal, and most signs of anemia were reversed. Liver 59Fe was elevated in CuD rats, but was restored to normal upon Cu repletion. These findings suggest a strong association between duodenal Hp abundance and Fe absorption in the CuD rat, and that reduced Fe absorption is an important factor in the cause of anemia.

Copper Deficiency Reduces Iron Absorption and Biological Half-Life in Male Rats

Dietary copper deficiency (CuD) in rats leads to iron (Fe) deficiency anemia. Is this because CuD reduces Fe absorption? Fe absorption in CuD rats was determined by feeding diets labeled with (59)Fe and using whole-body counting (WBC) to assess the amount retained over time. Two groups, each with 45 male weanling rats, were fed an AIN-93G diet low in Cu (<0.3 mg/kg; CuD) or one containing adequate Cu (5.0 mg/kg; CuA). At intervals over the next 42 d, 5 rats per group were killed and blood was drawn to determine hematocrit, hemoglobin, and other indicators of Fe status. At d 7 and 25, 5 rats per group were fed 1.0 g of their respective diets that had been labeled with (59)Fe. Retained (59)Fe was monitored for 10 d by WBC; then rats were killed and (59)Fe was measured in various organs. Signs of Fe deficiency, such as low hemoglobin, hematocrit, and RBC count, were evident in CuD rats by d 14. At d 7, CuD rats absorbed 90% as much Fe as CuA rats (P > 0.20), but at d 25, CuD rats absorbed only 50% as much as CuA rats (P < 0.001). In the study beginning at d 7, the biological half-life (BHL) of (59)Fe in CuD rats was less (P < 0.02) than that in CuA rats [geometric mean (-SEM, +SEM); 75(62,91) d vs. 175(156,195) d]. In the study beginning at d 25, the BHL was again less (P < 0.02) in the CuD rats than in the CuA rats [33(23,49) d for CuD and 157(148,166) d for CuA]. Apparently, the route of Fe loss in the CuD rats was through the gut. At d 16 and 34, CuD rats lost 4 to 5 times more (P < 0.01) (59)Fe in the feces in a 24-h period than the CuA rats. Also, (59)Fe in the duodenal mucosa of CuD rats was approximately 100% higher (P < 0.01) than in CuA rats. These findings suggest that Fe deficiency anemia in CuD male rats is caused at least in part by reductions in Fe absorption and retention.

Endothelial cell calcium mobilization to acetylcholine is attenuated in copper-deficient rats.

Dietary copper deficiency significantly attenuates nitric oxide (NO)-mediated vascular smooth muscle relaxation and vasodilation. There is evidence for both increased inactivation of the NO radical by superoxide anion, and oxidative damage to the endothelium where NO is produced. The current study was designed to examine the NO synthetic pathway in the endothelium during copper deficiency. Male weanling rats were fed a copper-adequate (CuA, 6.4 mg Cu/kg diet) or copper-deficient (CuD, 0.4 mg Cu/kg diet) diet for four weeks. Cremasteric arterioles (approximately 100 microm diameter) were isolated and used for the experiments. Western blot analysis of the arteriole endothelial nitric oxide synthase (eNOS) concentration did not show a difference between dietary groups. Acetylcholine (Ach)-induced vasodilation was significantly reduced in the CuD group both before and after pretreatment with the eNOS substrate L-arginine. Endothelial intracellular calcium ([Ca2+]i) stimulated by 10(-6) M Ach was significantly inhibited in the arterioles from CuD rats. Coincident with the inhibition of [Ca2+]i and vasodilation was a depression of vascular Cu/Zn-SOD activity and an increase in plasma peroxynitrite activity. These data suggest that endothelial Ca2+ signaling and agonist-stimulated NO-mediated vascular dilation are likely reduced by increased oxidative damage in copper-deficient rats.

Early and advanced glycation end-products are increased in dietary copper deficiency

The hypothesis that nonenzymatic glycosylation of proteins (glycation) contributes to damage associated with dietary copper deficiency has depended largely on indirect evidence. Thus far, the observation of an elevated percentage of glycated hemoglobin in copper-deficient rats has provided the only direct evidence of an increase in glycation. We sought further direct evidence of increased glycation in copper deficiency. Male weanling rats were fed a copper-adequate (CuA, 6.4 mg Cu/kg diet) or copper-deficient diet (CuD, 0.4 mg Cu/kg diet) for 5 weeks. Rats fed the CuD diet were copper deficient as judged by depressed organ copper concentrations and a variety of indirect indices. Measurements of hemoglobin A 1 and serum fructosamine (both early glycation end-products) as well as serum pentosidine (an advanced glycation end-product) indicated that all three compounds were elevated in CuD rats relative to CuA rats. This finding further supports the view that glycation is enhanced and thus may contribute to defects associated with dietary copper deficiency.

In vitro platelet adhesion to endothelial cells at low shear rates during copper deficiency in rats

Dietary copper restriction impairs thrombogenesis and hemostasis in rat microcirculation. In this study, the role of wall shear rate in platelet-to-endothelial cell adhesion in vitro was studied during copper deficiency. Platelets were obtained from male, weanling Sprague-Dawley rats fed purified diets, which were either copper-adequate (CuA, 6.3 μg Cu/g) or copper-deficient (CuD, 0.3 μg Cu/g), for 4 weeks. Platelets were through a parallel plate flow chamber containing cultured rat endothelial cells which were either normal or treated with the copper chelator tetraethylenepentamine (TEPA). Since platelet von Willebrand factor (vWF) concentration is decreased in CuD rats, we determined the platelet adhesion when CuD platelets were incubated in purified vWF (0.2 units/ml). Adhesion to normal endothelial cells was significantly lower for CuD platelets vs. CuA platelets at low (70 s−1; 21.5 ± 4.0% vs. 37.0 ± 2.7%) and relatively high (200 s−1; 9.3 ± 1.7% vs. 19.5 ± 1.1%) wall shear rates. Adhesion of CuD platelets to normal endothelial cells incubated in vWF was not different from adhesion of CuA platelets. Adhesion to TEPA-treated endothelial cells was lower than adhesion to normal endothelial cells for both CuA and CuD platelets (30.6 ± 1.5% vs. 37 ± 2.7%). Although increasing the shear rate (from 70 s−1 to 200 s−1) decreased adhesion of both CuA and CuD platelets, the ratio between groups remained similar. These results demonstrate that adhesion of CuD platelets to normal endothelial cells is less than that of CuA platelets under flow conditions typical for venules. Further, altered shear rate does not account for the depressed in vitro platelet adhesion. Thus, alteration of platelet and endothelial cell properties by copper deficiency may be of greater importance than the effect of flow conditions on endothelial cells in delaying thrombosis. J. Trace Elem. Exp. Med. 12:25–36, 1999. Published 1999 Wiley-Liss, Inc.

Hepatic fatty acid synthase gene transcription is induced by a dietary copper deficiency.

A dietary copper (Cu) deficiency is associated with a twofold increase in hepatic fatty acid biosynthesis. We hypothesized that the induction of hepatic lipogenesis associated with a dietary Cu deficiency reflected an enhanced expression of genes encoding lipogenic enzymes, i.e., fatty acid synthase (FAS). Male weanling rats were pair-meal fed for 42 days a high-sucrose diet that was Cu deficient (CuD; 0.7 microgram Cu/g) or Cu adequate (CuA; 5.0 micrograms Cu/g). The CuD diet increased FAS enzymatic activity twofold (P < 0.05). This rise in enzymatic activity was accompanied by a threefold increase in FAS mRNA and a 2.5-fold increase in FAS gene transcription (P < 0.05). Neither the mRNA abundance nor the rate of gene transcription for phosphoenolpyruvate carboxykinase or beta-actin was affected by the CuD diet. The induction of FAS gene transcription was associated with a 65-85% increase in hepatic reduced glutathione (GSH; P < 0.05). When hepatic GSH synthesis was suppressed by treating CuD rats with L-buthionine sulfoximine, the induction of FAS expression was completely prevented. Similarly, feeding N-acetylcysteine to CuA rats increased hepatic GSH levels 2.5-fold, and this was accompanied by a significant induction in FAS expression. These data indicate that the increase in hepatic lipogenesis associated with a Cu deficiency reflects an induction in hepatic lipogenic gene transcription (i.e., FAS) and that the rate of gene transcription may be dependent on hepatic thiol redox.

Suppression of renal γ-glutamylcysteine synthetase expression in dietary copper deficiency

A dietary deficiency of copper (CuD) is associated with a 50–70% and a 2-fold increase in hepatic reduced glutathione (GSH) concentration and synthesis, respectively, which leads to a 50–80% increase in plasma GSH. Moreover, the kidneys of CuD rats remove 40% more GSH from the blood than copper adequate (CuA) rats. These findings have led us to propose that the increase in hepatic synthesis of GSH in CuD rats is accompanied by a comparable increase in the hepatic expression of γ-glytamylcysteine synthetase (γ-GCS), the rate limiting enzyme of glutathione biosynthesis, and that the enhanced uptake of GSH by the kidney would lead to a compensatory decrease in renal γ-GCS expression. In experiment I, male weanling rats (3–4 weeks) were ad libitum fed a CuD (0.5 μg Cu/g) or CuA (5.8 μg/g) diet for 70 days; and in experiment II, male weanling rats were pair-meal fed the CuD or CuA diet for 35 days. In both studies, CuD diet caused a significant increase in hepatic GSH concentration, but hepatic γ-GCS activity and mRNA abundance were unchanged. In contrast, renal GSH concentration was unaffected by the CuD diet. However, renal γ-GCS activity was reduced 40% and this was paralleled by a 50% decrease in γ-GCS mRNA. Moreover, the decrease in renal γ-GCS mRNA was caused by a reduction in renal γ-GCS gene transcription. The results of these studies indicate that the increase in renal uptake of GSH resulting from a dietary Cu deficiency is associated with a compensatory decrease in γ-GCS expression.

Platelet Aggregation and Adhesion during Dietary Copper Deficiency in Rats

The role of dietary copper deficiency in platelet-to-endothelial cell adhesion and in platelet-to-platelet aggregation was studied in vitro. Platelets were obtained from male, weanling Sprague-Dawley rats fed purified diets which were either copper-adequate (CuA, 6.3 micrograms copper/g of diet) or copper-deficient (CuD, 0.3 microgram/g of diet) for 4 weeks. The platelet adhesion study was performed by adding CuA or CuD platelets either suspended in homologous plasma or in Tyrode buffer salt solution (TBSS) to cultured rate endothelial cells. After a one hour incubation at 37 degrees C non-adhered platelets were removed and counted in a microcytometer. Platelet aggregation in platelet rich plasma (PRP) samples was induced by adding ADP (2 x 10(-4)M) and measured in a turbidometric aggregometer. The content of von Willebrand factor (vWF) in platelets and in plasma and the content of fibrinogen in platelets was determined. Platelet adhesion to rat endothelial cells was significantly lower for platelets from CuD rats than for platelets from CuA rats. ADP induced platelet aggregation from CuD rats was significantly higher than platelet aggregation from CuA rats. The content of vWF in platelets and in plasma from CuD rats was significantly lower than in platelets and plasma from CuA rats. However, the amount of fibrinogen in platelets from ++CuD rats was about 4-fold higher than that in platelets from CuA rats while the plasma fibrinogen was lower in CuD rats than in CuA rats. These studies illustrate that copper deficiency diminishes platelet adhesion to endothelial cells but increases platelet aggregability. The results suggest that these physiological alterations may be the result of decreased platelet vWF and increased platelet fibrinogen during dietary copper deficiency.

Dietary copper deficiency reduces heat shock protein expression in cardiovascular tissues

Dietary copper deficiency impairs cardiovascular function by depression of catecholamine metabolism, and alteration of the structure and function of cardiac mitochondria. Heat shock proteins (HSPs) are a group of cellular homeostatic proteins that are induced in vascular tissue by catecholaminergic transmission after exposure to stress. We investigated the effects of dietary copper deficiency on the induction and accumulation of HSPs in several cardiovascular tissues. Stress-induced levels of aortic HSP70 mRNA were reduced in copper-deficient (CuD) rats when compared with copper-adequate (CuA) controls. Cocaine-induced HSP70 mRNA accumulation was not different between CuA and CuD rats, suggesting that reduced HSP70 levels in restrained CuD animals may result from altered catecholaminergic neurotransmission. The level of HSP60 mRNA was specifically reduced in the atria of CuD rats, which may be associated with altered mitochondrial structure and function. These results describe a novel relationship between dietary copper deficiency and the expression of highly conserved cellular stress response proteins. Loss of these essential homeostatic proteins in vascular tissue may contribute to the impairment of cardiovascular function known to accompany copper deficiency.