Effects of prenatal low protein and postnatal high fat diets on visceral adipose tissue macrophage phenotypes and IL-6 expression in Sprague Dawley rat offspring.

Adipose tissue macrophages (ATM) are implicated in adipose tissue inflammation and obesity-related insulin resistance. Maternal low protein models result in fetal programming of obesity. The study aims to answer whether maternal undernutrition by protein restriction affects the ATM M1 or M2 phenotype under postnatal high fat diet in F1 offspring. Using a rat model of prenatal low protein (LP, 8% protein) diet followed by a postnatal high fat energy diet (HE, 45% fat) or low fat normal energy diet (NE, 10% fat) for 12 weeks, we investigated the effects of these diets on adiposity, programming of the offspring ATM phenotype, and the associated inflammatory response in adipose tissue. Fat mass in newborn and 12-week old LP fed offspring was lower than that of normal protein (20%; NP) fed offspring; however, the adipose tissue growth rate was higher compared to the NP fed offspring. While LP did not affect the number of CD68+ or CD206+ cells in adipose tissue of NE offspring, it attenuated the number of these cells in offspring fed HE. In offspring fed HE, LP offspring had a lower percentage of CD11c+CD206+ ATMs, whose abundancy was correlated with the size of the adipocytes. Noteworthy, similar to HE treatment, LP increased gene expression of IL-6 within ATMs. Two-way ANOVA showed an interaction of prenatal LP and postnatal HE on IL-6 and IL-1ß transcription. Overall, both LP and HE diets impact ATM phenotype by affecting the ratio of CD11c+CD206+ ATMs and the expression of IL-6.

Skeletal muscle Sirt3 expression and mitochondrial respiration are regulated by a prenatal low-protein diet.

Malnutrition during the fetal growth period increases risk for later obesity and type 2 diabetes mellitus (T2DM). We have shown that a prenatal low-protein (8% protein; LP) diet followed by postnatal high-fat (45% fat; HF) diet results in offspring propensity for adipose tissue catch-up growth, obesity and T2DM in Sprague-Dawley rats. Skeletal muscle is the major tissue for insulin-mediated glucose uptake. Dysfunctional skeletal muscle mitochondrial function, particularly reduction in expression of mitochondrial protein sirtuin protein 3 (Sirt3) contributes to development of T2DM by reducing mitochondrial respiration. Therefore, we hypothesized that maternal LP and postnatal HF diets would increase T2DM risk due to Sirt3 dysfunction within skeletal muscle mitochondria. Using our maternal LP and postnatal HF diet model, we showed that skeletal muscle mitochondrial oxygen consumption rate was decreased by maternal LP diet. Mitochondria copy number, mitochondrial thermogenesis (UCP-1) expression and mitochondrial biogenic factors including nuclear respiratory factor 1 and cytochrome c oxidases 1 and 4 were unaffected by maternal LP and postnatal HF diets. Skeletal muscle Sirt3 mRNA decreased with maternal LP diet. A mitochondrial substrate of Sirt3, succinate dehydrogenase (SDH), is regulated by Sirt3 via lysine residue acetylation status of SDH. Acetylated SDH protein (inactive form) levels were moderately decreased by maternal LP diet. Taken together, these data suggest that maternal LP and postnatal HF diets may increase the risk for T2D by decreasing skeletal muscle oxidative respiration via increased Sirt3 and possibly by decreased amounts of the active form of SDH enzyme.

Prenatal low-protein and postnatal high-fat diets induce rapid adipose tissue growth by inducing Igf2 expression in Sprague Dawley rat offspring.

Maternal low-protein diets result in lower birth weight followed by accelerated catch-up growth that is accompanied by the development of obesity and glucose intolerance in later life. Whether postnatal high-fat (HF) diets further contribute to the development of obesity and insulin resistance in offspring by affecting adipose tissue metabolism and DNA methylation is currently unknown. Obese-prone Sprague-Dawley rats were fed 8% low protein (LP) or 20% normal protein diets for 3 wk prior to conception and throughout pregnancy and lactation to investigate whether prenatal LP and postnatal HF diets affect the rate of adipose tissue growth, insulin-like growth factor 2 (Igf2) expression, and DNA methylation in male offspring. At weaning, the offspring were fed 10% normal fat or 45% HF diets for 12 wk. The adipose tissue growth rate was increased (up to 26-fold) by the LP prenatal and HF postnatal diets. Adipose tissue Igf2 mRNAs and DNA methylation were increased by the LP prenatal and HF postnatal diets. The LP prenatal and HF postnatal diet increased the number of small adipocytes in adipose tissue and decreased insulin sensitivity. These findings suggest that prenatal LP and postnatal HF intake result in adipose tissue catch-up growth through alterations in the expression of the Igf2 gene and DNA methylation within adipocytes. These alterations in adiposity are accompanied by an increased risk of development of type 2 diabetes.

Modest fructose beverage intake causes liver injury and fat accumulation in marginal copper deficient rats.

OBJECTIVE: Dietary fructose and copper interaction may play an important role in the pathogenesis of nonalcoholic fatty liver disease. In this study, whether or not modest fructose consumption (3% fructose, w/v) (which is more closely related to the American lifestyle with regard to sugar beverage consumption) affects copper status, and causes liver injury and fat accumulation in marginal copper deficient rats was investigated. DESIGN AND METHODS: Male weanling Sprague-Dawley rats were fed either an adequate copper (6 ppm) or a marginally copper deficient (1.6 ppm) diet for 4 weeks. Deionized water or deionized water containing 3% fructose (w/v) was given ad lib. RESULTS: Modest fructose consumption further impaired copper status in the marginal copper deficient rats and increased hepatic iron accumulation. Liver injury and fat accumulation were significantly induced in the marginal copper deficient rats exposed to fructose. CONCLUSIONS: Our data suggest that modest fructose consumption can impair copper status and lead to hepatic iron overload, which in turn, may lead to liver injury and fatty liver in marginal copper deficient rats. This study provides important information on dietary fructose and copper interaction, suggesting that dietary fructose-induced low copper availability might be an important mechanism underlying fructose-induced fatty liver.

High fructose feeding induces copper deficiency in Sprague-Dawley rats: a novel mechanism for obesity related fatty liver.

BACKGROUND & AIMS: Dietary copper deficiency is associated with a variety of manifestations of the metabolic syndrome, including hyperlipidemia and fatty liver. Fructose feeding has been reported to exacerbate complications of copper deficiency. In this study, we investigated whether copper deficiency plays a role in fructose-induced fatty liver and explored the potential underlying mechanism(s). METHODS: Male weanling Sprague-Dawley rats were fed either an adequate copper or a marginally copper deficient diet for 4 weeks. Deionized water or deionized water containing 30% fructose (w/v) was also given ad lib. Copper and iron status, hepatic injury and steatosis, and duodenum copper transporter-1 (Ctr-1) were assessed. RESULTS: Fructose feeding further impaired copper status and led to iron overload. Liver injury and fat accumulation were significantly induced in marginal copper deficient rats exposed to fructose as evidenced by robustly increased plasma aspartate aminotransferase (AST) and hepatic triglyceride. Hepatic carnitine palmitoyl-CoA transferase I (CPT I) expression was significantly inhibited, whereas hepatic fatty acid synthase (FAS) was markedly up-regulated in marginal copper deficient rats fed with fructose. Hepatic antioxidant defense system was suppressed and lipid peroxidation was increased by marginal copper deficiency and fructose feeding. Moreover, duodenum Ctr-1 expression was significantly increased by marginal copper deficiency, whereas this increase was abrogated by fructose feeding. CONCLUSIONS: Our data suggest that high fructose-induced nonalcoholic fatty liver disease (NAFLD) may be due, in part, to inadequate dietary copper. Impaired duodenum Ctr-1 expression seen in fructose feeding may lead to decreased copper absorption, and subsequent copper deficiency.

Expression of oxidative phosphorylation components in mitochondria of long-living Ames dwarf mice.

Reduced signaling of the growth hormone (GH)/insulin-like growth factor-1 (IGF-1) pathway is associated with extended life span in several species. Ames dwarf mice are GH-deficient and live >50% longer than wild-type littermates. Previously, we have shown that tissues from Ames mice exhibit elevated levels of antioxidative enzymes, less H(2)O(2) production, and lower oxidative damage suggesting that mitochondrial function may differ between genotypes. To explore the relationship between hormone deficiency and mitochondria in mice with extended longevity, we evaluated activity, protein, and gene expression of oxidative phosphorylation components in dwarf and wild-type mice at varying ages. Liver complex I + III activity was higher in dwarf mice compared to wild-type mice. The activity of I + III decreased between 3 and 20 months of age in both genotypes with greater declines in wild-type mice in liver and skeletal muscle. Complex IV activities in the kidney were elevated in 3- and 20-month-old dwarf mice relative to wild-type mice. In Ames mice, protein levels of the 39 kDa complex I subunit were elevated at 20 months of age when compared to wild-type mouse mitochondria for every tissue examined. Kidney and liver mitochondria from 20-month-old dwarf mice had elevated levels of both mitochondrially-encoded and nuclear-encoded complex IV proteins compared to wild-type mice (p < 0.05). Higher liver ANT1 and PGC-1α mRNA levels were also observed in dwarf mice. Overall, we found that several components of the oxidative phosphorylation (OXPHOS) system were elevated in Ames mice. Mitochondrial to nuclear DNA ratios were not different between genotypes despite the marked increase in PGC-1α levels in dwarf mice. The increased OXPHOS activities, along with lower ROS production in dwarf mice, predict enhanced mitochondrial function and efficiency, two factors likely contributing to long-life in Ames mice.

Parasite infection is associated with Kaposi's sarcoma associated herpesvirus (KSHV) in Ugandan women.

BACKGROUND: Immune modulation by parasites may influence susceptibility to bacteria and viruses. We examined the association between current parasite infections, HIV and syphilis (measured in blood or stool samples using standard methods) and antibodies against Kaposi's sarcoma herpesvirus (KSHV), measured by ELISA, in 1915 stored plasma samples from pregnant women in Entebbe, Uganda. RESULTS: Seroprevalence of KSHV was higher in women with malaria parasitaemia (73% vs 60% p = 0.01), hookworm (67% vs 56% p = 0.001) and Mansonella perstans (69% vs 59% p = 0.05); seroprevalence increased with increasing intensity of hookworm infection (p < 0.001[trend]). No associations were found for HIV, five other parasites or active syphilis. These effects were not explained by socioeconomic status or education. CONCLUSIONS: Specific parasite infections are associated with presence of antibodies against KSHV, perhaps mediated via their effect on immune function.

Cyclooxygenase-2 is upregulated in copper-deficient rats.

Copper deficiency inactivates Cu/Zn-SOD and promotes accumulation of reactive oxygen species. This process likely impairs nitric oxide (NO)-mediated relaxation as well as triggers vascular inflammation. The current study was designed to determine whether COX-2, a proinflammatory protein, expression and activity are upregulated in the oxidative environment associated with inadequate Cu. Weanling male Sprague Dawley rats were fed purified diets which were either Cu-adequate (Cu-A); Cu-marginal (Cu-M), Cu-deficient (Cu-D), or the Cu-D diet combined with the SOD mimetic Tempol (Cu-D/T; 1 mM in drinking water) for 4 weeks. COX-2 protein, PGE(2) (COX-2 metabolite) and isoprostanes (index of oxidative stress) were all higher in the Cu-D group vs Cu-A group, but no significant differences occurred between the Cu-M and Cu-A groups. Tempol protected against an attenuation of NO-mediated vasodilation in the Cu-D rats but did not prevent the elevation of PGE(2) or isoprostanes. Our data suggest a role for copper as a modulator of oxidative stress and inflammation independent of SOD activity or NO-derived oxidants.

Copper deficiency inhibits Ca2+-induced swelling in rat cardiac mitochondria.

Cu deficiency disrupts the architecture of mitochondria, impairs respiration, and inhibits the activity of cytochrome c oxidase - the terminal, Cu-dependent respiratory complex (Complex IV) of the electron transport chain. This suggests that perturbations in the respiratory chain may contribute to the changes in mitochondrial structure caused by Cu deficiency. This study investigates the effect of Cu deficiency on Ca2+-induced mitochondrial swelling as it relates to changes in respiratory complex activities in cardiac mitochondria of rats. Male weanling rats were fed diets containing either no added Cu (Cu0), 1.5 mg Cu/kg (Cu1.5), 3 mg Cu/kg (Cu3) or 6 mg Cu/kg (Cu6). The rate of Ca2+-induced mitochondrial swelling in the presence of succinate and oligomycin was reduced, and the time to reach maximal swelling was increased only in the rats consuming Cu0 diet. Cytochrome c oxidase activity was reduced 60% and 30% in rats fed Cu0 and Cu1.5, respectively, while NADH:cytochrome c reductase (Complex I+ComplexIII) activity was reduced 30% in rats consuming both Cu0 and Cu1.5. Mitochondrial swelling is representative of mitochondrial permeability transition pore (MPTP) formation and the results suggest that Ca2+-induced MPTP formation occurs in cardiac mitochondria of Cu-deficient rats only when cytochrome c oxidase activity falls below 30% of normal. Decreased respiratory complex activities caused by severe Cu deficiency may inhibit MPTP formation by increasing matrix ADP concentration or promoting oxidative modifications that reduce the sensitivity of the calcium trigger for MPTP formation.

Regression of copper-deficient heart hypertrophy: reduction in the size of hypertrophic cardiomyocytes.

Dietary copper (Cu) deficiency causes cardiac hypertrophy and its transition to heart failure in a mouse model. Cu repletion results in rapid regression of cardiac hypertrophy and prevention of heart failure. The present study was undertaken to understand dynamic changes of cardiomyocytes in the hypertrophic heart during the regression. Dams of FVB mice were fed a Cu-deficient (CuD) diet (0.3 mg Cu/kg) starting on Day 3 post-delivery, and weanling pups were fed the same diet until Cu repletion (6.0 mg Cu/kg) in the diet at 31 days of age. Heart samples were obtained at the end of CuD feeding or at 3, 7, 14 or 28 days after Cu repletion. Cu deficiency resulted in increases in the size and reduction in the number of cardiomyocytes in the heart. Cu repletion led to regression in the size of hypertrophic cardiomyocytes and normalization of the total number of cardiomyocytes. Although a direct reduction in the cell size would be significantly responsible for the regression of heart hypertrophy, some hypertrophic cardiomyocytes upon Cu repletion reentered the cell cycle as determined by Ki-67 staining in the cardiomyocyte-specific alpha-sarcomeric actin-stained cells and underwent division as determined by a mitosis-specific marker, phospho-histone 3. Quantitative analysis indicated that the replication of hypertrophic cardiomyocytes made a contribution of about one-third to the total mitosis of the regenerated myocardium. This study suggests that a direct reduction in the size of some hypertrophic cardiomyocytes and a replication of other hypertrophic cardiomyocytes with reduced size make a significant contribution to the regression of CuD heart hypertrophy, leading to normalization of the size and the number of cardiomyocytes in the heart.

Endothelial cell-derived nitric oxide mobilization is attenuated in copper-deficient rats.

The attenuation of endothelium-dependent nitric oxide (NO) mediated vasodilation is a consistent finding in both conduit and resistance vessels during dietary copper (Cu) deficiency. Although the effect is well established, evidence for the mechanism remains circumstantial. This study was designed to determine the relative amount of NO produced in and released from the vascular endothelium. Using the fluorescent NO indicator, 4-amino-5-methylamino-2',7'-difluorofluorescein (DAF-FM), we now demonstrate the effect of a Cu-deficient diet on the production of NO from the endothelium of resistance arterioles. In one group of experiments, control and Cu-chelated lung microvascular endothelial cells (ECs) were used to assay NO production and fluorescence was observed by confocal microscopy. Weanling Sprague-Dawley rats were fed purified diets that were either Cu adequate (6.3 micrograms Cu per gram of food) or Cu deficient (0.3 micrograms Cu per gram of food) for 4 weeks. In the second series of experiments, first-order arterioles were microsurgically isolated from the rat cremaster muscle, cannulated, and pressurized with (3[N-morpholino]propanesulfonic acid) physiologic salt solution (MOPS-PSS). DAF-FM (5 micromol.L-1) was added in the lumen of the vessel to measure NO release. Baseline DAF-FM fluorescence was significantly lower in Cu-chelated ECs than in controls. In response to 10-6 mol.L-1 acetylcholine, fluorescent intensity was significantly less in chelated ECs and in the lumen of Cu-deficient arterioles. The results suggest that production and release of NO by the vascular endothelium is inhibited by a restriction of Cu. This inhibition may account for the attenuated vasodilation previously reported in Cu-deficient rats.

Cardiac cytochrome C oxidase activity and contents of subunits 1 and 4 are altered in offspring by low prenatal copper intake by rat dams.

It has been reported previously that the offspring of rat dams consuming low dietary copper (Cu) during pregnancy and lactation experience a deficiency in cardiac cytochrome c oxidase (CCO) characterized by reduced catalytic activity and mitochondrial and nuclear subunit content after postnatal d 10. The present study was undertaken to determine whether the cardiac CCO deficiency was caused directly by low postnatal Cu intake or whether it was a prenatal effect of low Cu intake by the dams that became manifest postnatally. Dams were fed either a Cu-adequate diet (6 mg Cu/kg) or Cu-deficient diet (1 mg Cu/kg) beginning 3 wk before conception and throughout gestation and lactation. One day following parturition, several litters from Cu-adequate dams were cross fostered to Cu-deficient dams and several litters from Cu-deficient dams were cross fostered to Cu-adequate dams. Litters that remained with their birth dams served as controls. CCO activity, the content of the mitochondrial-encoded CCO subunit 1 (COX1), and the content of the nuclear-encoded subunit COX4 in cardiac mitochondria were reduced in the 21-d-old offspring of Cu-deficient dams. COX1 content was normal in the 21-d-old cross-fostered offspring of Cu-deficient dams, but CCO activity and COX4 were reduced. Cross fostering the offspring of Cu-adequate dams to Cu-deficient dams did not significantly affect CCO activity, COX1 content, or COX4 content in cardiac mitochondria of 21-d-old offspring. These data indicate that low prenatal Cu intake by dams was the determinant of CCO activity in cardiac mitochondria of the 21-d-old offspring and may have led to the assembly of a less-than-fully active holoenzyme.

Dietary interaction of high fat and marginal copper deficiency on cardiac contractile function.

OBJECTIVE: High-fat and marginally copper-deficient diets impair heart function, leading to cardiac hypertrophy, increased lipid droplet volume, and compromised contractile function, resembling lipotoxic cardiac dysfunction. However, the combined effect of the two on cardiac function is unknown. This study was designed to examine the interaction between high-fat and marginally copper-deficient diets on cardiomyocyte contractile function. RESEARCH METHODS AND PROCEDURES: Weanling male rats were fed diets incorporating a low- or high-fat diet (10% or 45% of kcal from fat, respectively) with adequate (6 mg/kg) or marginally deficient (1.5 mg/kg) copper content for 12 weeks. Contractile function was determined with an IonOptix system including peak shortening (PS), time-to-PS, time-to-90% relengthening, maximal velocity of shortening/relengthening, and intracellular Ca(2+) ([Ca(2+)](I)) rise and decay. RESULTS: Neither dietary treatment affected blood pressure or glucose levels, although the high-fat diet elicited obesity and glucose intolerance. Both diets depressed PS, maximal velocity of shortening/relengthening, and intracellular Ca(2+) ([Ca(2+)](I)) rise and prolonged time-to-90% relengthening and Ca(2+) decay without an additive effect between the two. Ca(2+) sensitivity, apoptosis, lipid peroxidation, nitrosative damage, tissue ceramide, and triglyceride levels were unaffected by either diet or in combination. Phospholamban (PLB) but not sarco(endo)plasmic reticulum Ca(2+)-ATPase was increased by both diets. Endothelial NO synthase was depressed with concurrent treatments. The electron transport chain was unaffected, although mitochondrial aconitase activity was inhibited by the high-fat diet. DISCUSSION: These data suggest that high-fat and marginally copper deficient diets impaired cardiomyocyte contractile function and [Ca(2+)](i) homeostasis, possibly through a similar mechanism, without obvious lipotoxicity, nitrosative damage, and apoptosis.

Hearts in adult offspring of copper-deficient dams exhibit decreased cytochrome c oxidase activity, increased mitochondrial hydrogen peroxide generation and enhanced formation of intracellular residual bodies.

The long-term effects of low dietary copper (Cu) intake during pregnancy and lactation on cardiac mitochondria in first-generation adult rats was examined. Rat dams were fed diets containing either low (1 mg/kg Cu) or adequate (6 mg/kg Cu) levels of dietary Cu beginning 3 weeks before conception and ending 3 weeks after birth. Cytochrome c oxidase (CCO) activity was 51% lower in isolated cardiac mitochondria from 21-day-old offspring of Cu-deficient dams than in the offspring of Cu-adequate dams. CCO activities in the cardiac mitochondria of 63- and 290-day-old offspring were 22% lower and 14% lower, respectively, in the offspring of Cu-deficient dams after they had been repleted with adequate dietary Cu from the time they were 21 days old. Electron micrographs showed that the size of residual bodies and the cellular volume they occupied in cardiomyocytes rose significantly between 63 and 290 days in the Cu-repleted offspring of Cu-deficient dams, but not in the offspring of Cu-adequate dams. The rate of hydrogen peroxide generation by cardiac mitochondria also was 24% higher in the 290-day-old repleted offspring of Cu-deficient dams than in the offspring of Cu-adequate dams. The increase in hydrogen peroxide production by cardiac mitochondria and in the relative volume and size of dense deposits in cardiomyocytes is consistent with increased oxidative stress and damage resulting from prolonged reduction of CCO activity in the offspring of Cu-deficient dams.

Copper deficiency decreases complex IV but not complex I, II, III, or V in the mitochondrial respiratory chain in rat heart.

It has been documented that dietary copper (Cu) deficiency impairs mitochondrial respiratory function, which is catalyzed by 5 membrane-bound multiple protein complexes. However, there are few reports on the simultaneous analysis of Cu effect on the subunit protein expression on all 5 protein complexes. The present study was undertaken to determine the effect of Cu deficiency on each mitochondrial respiratory complex's protein expression in rat heart tissue with western-blot analysis. Male Sprague-Dawley rats were fed diets that were either Cu adequate (6.0 microg Cu/g diet, n = 5) or Cu deficient (0.3 microg Cu/g diet, n = 5) for 5 wk. The monoclonal antibody-based western-blot analysis suggested that the protein levels of 39-kDa and 30-kDa subunits in complex I; 70-kDa and 30-kDa subunits in complex II; core I and core II subunits in complex III; and alpha and beta subunits of F1 complex in complex V in both high-salt buffer (HSB) and low-salt buffer (LSB) protein fractions from heart tissue of Cu-deficient rats did not differ from those of Cu-adequate rats. However, the protein level of cytochrome c oxidase (CCO) subunit (COX) I, COX Vb, and COX VIb subunits in complex IV (CCO) in both HSB and LSB protein fractions from heart tissue of Cu-deficient rats was lower than those of Cu-adequate rats. Collectively, these data demonstrate that Cu deficiency decreases each tested subunit protein expression of complex IV but not those of complex I, II, III, and V in mitochondrial respiratory complexes.

Pinto beans are a source of highly bioavailable copper in rats.

The trace element copper (Cu) is a required nutrient in the diets of humans. It has been found in animal studies to be essential for efficient iron absorption and oxygen utilization and for aiding free-radical degradation. Dry beans (Phaseolis vulgaris) are potentially good sources of Cu; thus, the objective of this study was to determine the bioavailability of Cu from dry beans using the pinto bean as the source. Dry beans were obtained from a local market, cooked according to package directions, and dried. Weanling male Sprague-Dawley rats (8 groups of 8 rats each) were fed a Cu-deficient diet (AIN-93G) for 4 wk followed by 2 wk of Cu repletion with diets containing 0-6.5 mg Cu/kg diet added as CuSO(4) or with 0.6 and 1.5 mg Cu/kg incorporated into rat diets as pinto beans at 10 and 20%. Standard response curves were developed based on repletion-induced recovery of 10 indices of Cu status, including organ Cu concentrations and Cu-dependent enzyme activities, in response to increasing dietary Cu as CuSO(4). Recovery of these variables in rats fed the pinto bean diets was compared with the standard response curve at similar levels of dietary Cu. Based on the recovery of all 10 variables, the relative bioavailability of Cu from dry beans was at least 100% of that with the highly available CuSO(4). For 3 of the variables, liver and heart Cu concentrations and serum superoxide dismutase 3 activity, estimated bioavailability values of Cu from beans were 138, 140, and 134%, respectively, of those from CuSO(4). We conclude that the dry pinto bean is a good source of dietary Cu with respect to both concentration and bioavailability.

Cardiac cytochrome-c oxidase deficiency occurs during late postnatal development in progeny of copper-deficient rats.

Although cytochrome-c oxidase (CCO) is a copper-dependent enzyme, the effect of maternal copper deficiency on the expression of CCO activity during postnatal development of the neonatal rat heart has not been investigated extensively. Here, we show that CCO activity in heart mitochondria isolated from neonates of copper-deficient dams did not exhibit significant reductions until postnatal days (PND) 15 and 21. In addition, immunoblot analysis indicated that the CCO subunit (Cox-1) was reduced on postnatal Days 10 and 21, and that Cox-4 was reduced on PND 21 in heart mitochondria of the neonates from copper-deficient dams. These findings indicate that the impairment of CCO activity in neonatal heart by maternal copper deficiency occurs late in the postnatal heart development. Furthermore, the concurrent reductions in Cox-1 and Cox-4 suggest that the impaired CCO activity reflects a CCO deficiency in heart mitochondria. CCO activity and Cox-1 in heart mitochondria were not fully restored by 6 weeks of postweaning copper repletion in the pups of copper-deficient dams. This indicates that prolonged maternal intake of moderately low dietary copper produces CCO deficiency in cardiac mitochondria of neonates during late postnatal heart development, after terminal differentiation of cardiomyocytes occurs. The resistance of CCO deficiency to repair by dietary copper supplementation may be related to the relatively slow turnover of the affected mitochondria in the terminally differentiated heart.

High-fat diet-induced juvenile obesity leads to cardiomyocyte dysfunction and upregulation of Foxo3a transcription factor independent of lipotoxicity and apoptosis.

BACKGROUND: Obesity is associated with dyslipidemia, which leads to elevated triglyceride and ceramide levels, apoptosis and compromised cardiac function. METHODS: To determine the role of high-fat diet-induced obesity on cardiomyocyte function, weanling male Sprague-Dawley rats were fed diets incorporating 10% of kcal or 45% of kcal from fat. Mechanical function of ventricular myocytes was evaluated including peak shortening (PS), time-to-PS (TPS), time-to-90% relengthening (TR90) and maximal velocity of shortening and relengthening (+/- dl/dt). Intracellular Ca properties were assessed using fluorescent microscopy. RESULTS: High-fat diet induced hyperinsulinemic insulin-resistant obesity with depressed PS, +/- dl/dt, prolonged TPS/TR90 reduced intracellular Ca release and Ca clearing rate in the absence of hypertension, diabetes, lipotoxicity and apoptosis. Myocyte responsiveness to increased stimulus frequency and extracellular Ca was compromised. SERCA2a and phospholamban levels were increased, whereas phosphorylated phospholamban and potassium channel (Kv1,2) were reduced in high-fat diet group. High-fat diet upregulated the forkhead transcription factor Foxo3a, and suppressed mitochondrial aconitase activity without affecting expression of the caloric sensitive gene silent information regulator 2 (Sir2), protein nitrotyrosine formation, lipid peroxidation and apoptosis. Levels of endothelial nitric oxide synthase (NOS), inducible NOS, triglycerides and ceramide were similar between the two groups. CONCLUSIONS: Collectively, our data show that high-fat diet-induced obesity resulted in impaired cardiomyocyte function, upregulated Foxo3a transcription factor and mitochondrial damage without overt lipotoxicity or apoptosis.

Serum superoxide dismutase 3 (extracellular superoxide dismutase) activity is a sensitive indicator of Cu status in rats.

Sensitivity of the assay for Cu,Zn superoxide dismutase 3 (SOD3), the predominant form of SOD in serum, can be increased, and interferences caused by low-molecular-weight substances in the serum can be reduced by conducting the assay at pH 10 with xanthine/xanthine oxidase and acetylated cytochrome c (cyt c) as superoxide generator and detector, respectively. Serum SOD3 activity was assayed under these conditions in an experiment where weanling, male rats were fed diets for 6 weeks containing 3, 5 and 15 mg Zn/kg with dietary Cu set at 0.3, 1.5 and 5 mg Cu/kg at each level of dietary Zn. Serum SOD3 responded to changes in dietary Cu but not to changes in dietary Zn. A second experiment compared serum SOD3 activity to traditional indices of Cu status in weanling, male and female rats after they were fed diets containing, nominally, 0, 1, 1.5, 2, 2.5, 3 and 6 mg Cu/kg for 6 weeks. Serum SOD3 activity was significantly lower (P < .05) in male rats fed diets containing 0 and 1 mg Cu/kg and female rats fed diet containing 0 mg Cu/kg compared with rats fed diet containing 6 mg Cu/kg. These changes were similar to changes in liver Cu concentrations, liver cyt c oxidase (CCO) activity and plasma ceruloplasmin in males and females. Serum SOD3 activity was also strongly, positively correlated with liver Cu concentrations over the entire range of dietary Cu concentrations (R(2) = .942 in males, R(2) = .884 in females, P < .0001). Plots of serum SOD3 activity, liver Cu concentration, liver CCO activity and ceruloplasmin as functions of kidney Cu concentration all had two linear segments that intersected at similar kidney Cu concentrations (18-22 microg/g dry kidney in males, 15-17 microg/g dry kidney in females). These findings indicate that serum SOD3 activity is a sensitive index of Cu status.

Dietary copper deficiency reduces iron absorption and duodenal enterocyte hephaestin protein in male and female rats.

The mechanism for reduced Fe absorption in Cu deficiency is unknown, but may involve the intestinal Cu-dependent ferroxidase, Hephaestin (Hp). A 2 x 2 factorial experiment was designed to include Cu-deficient (CuD) and Cu-adequate (CuA) male and female rats. Weanling rats of both sexes were randomly divided into 2 groups each and fed an AIN-93G diet with low (<0.3 mg/kg; CuD) or adequate Cu (5.0 mg/kg; CuA). After 19 d, rats were fed 1.0 g each of their respective diets labeled with (59)Fe. Retained (59)Fe was monitored by whole-body counting for 12 d. Then, rats were killed for (59)Fe and Fe measurements in blood and various organs. Duodenal enterocytes were isolated for Western blot analysis of Hp. Signs of Cu and Fe deficiency were evident in both sexes. CuD male rats absorbed 60% as much Fe as CuA male rats (P < 0.001), whereas CuD female rats absorbed 70% (P < 0.001) as much as CuA females, with no difference between the sexes. Hp protein in enterocytes of CuD rats of both sexes was only 35% of that in CuA rats. The biological half-life of (59)Fe in CuD rats was only 50% (P < 0.001) of that in CuA rats, suggesting that Fe turnover was faster in CuD rats than CuA rats. Serum, spleen, and kidney Fe were lower (P < 0.001) in CuD rats than in CuA rats. Duodenal mucosa and liver Fe were higher (P < 0.01) in CuD male rats than CuA rats. Duodenal Fe but not liver Fe was higher in CuD female rats than CuA rats. Liver Fe was much higher (<0.001) overall in females than males. The data suggest that Cu deficiency reduces Fe absorption in rats through reduced expression of duodenal Hp protein.