W. Thomas Johnson

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 Ca2+ 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 Ca2+ release and Ca2+ clearing rate in the absence of hypertension, diabetes, lipotoxicity and apoptosis. Myocyte responsiveness to increased stimulus frequency and extracellular Ca2+ 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.

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.

Mitochondrial oxidant generation and oxidative damage in Ames dwarf and GH transgenic mice

Aging is associated with an accumulation of oxidative damage to proteins, lipids and DNA. Cellular mechanisms designed to prevent oxidative damage decline with aging and in diseases associated with aging. A long-lived mouse, the Ames dwarf, exhibits growth hormone deficiency and heightened antioxidative defenses. In contrast, animals that over express GH have suppressed antioxidative capacity and live half as long as wild type mice. In this study, we examined the generation of H2O2 from liver mitochondria of Ames dwarf and wild type mice and determined the level of oxidative damage to proteins, lipids and DNA in various tissues of these animals. Dwarf liver mitochondria (24 months) produced less H2O2 than normal liver in the presence of succinate (p<0.03) and ADP (p<0.003). Levels of oxidative DNA damage (8ÕHdG) were variable and dependent on tissue and age in dwarf and normal mice. Forty-seven percent fewer protein carbonyls were detected in 24-month old dwarf liver tissue compared to controls (p<0.04). Forty percent more (p<0.04) protein carbonyls were detected in liver tissue (3-month old) of GH transgenic mice compared to wild types while 12 month old brain tissue had 53% more protein carbonyls compared to controls (p<0.005). Levels of liver malonaldehyde (lipid peroxidation) were not different at 3 and 12 months of age but were greater in Ames dwarf mice at 24 months compared to normal mice. Previous studies indicate a strong negative correlation between plasma GH levels and antioxidative defense. Taken together, these studies show that altered GH-signaling may contribute to differences in the generation of reactive oxygen species, the ability to counter oxidative stress and life span.

Platelet cytochrome C oxidase activity is an indicator of copper status in rats

Abstract Male weanling rats were fed diets containing 0.2, 1.0, 1.9, 3.0, 4.2 and 5.4 μg Cu/g for five weeks. Significant (P 2 =0.83) and hepatic Cu,Zn-superoxide dismutase activity (R 2 =0.78). Also, inclusion of platelet cytochrome c oxidase activity with ceruloplasmin activity and erythrocyte superoxide dismutase activity improved the ability of these indices to predict liver copper concentration and liver Cu,Zn-superoxide dismutase activity. These findings indicate that platelet cytochrome c oxidase activity may be useful as an indictor of copper status, particularly in marginal status and when used with one or more other indices of copper status.

Lymphoid cell functions during copper deficiency

Abstract Male weanling Lewis rats were fed diets either adequate (5.6 μg/g) or low (0.6 μg/g) in copper, either and libitum or in restricted amounts for 14–70 days. Measurements were made of food intake; growth rate; hematocrits; Cu and Fe concentrations of plasma or serum; ceruloplasmin activity of plasma or serum; plasma cholesterol and whole blood lactate concentrations; cytochrome c oxidase activity of spleen lymphoid cells (SLC); and proliferation of SLC, cervical lymph node cells (CLNC) and plastic nonadherent-SLC (NASLC) following stimulation with concanavalin-A (Con-A). Cu-deficient (CD) rats exhibited upon completion of dietary regimen days: 14–18, reduced hematocrits, plasma Cu and Fe, plasma ceruloplasmin activity and cytochrome c oxidase activity of SLC; 28–32, elevated plasma cholesterol and blood lactate, decreased proliferation of Con-A stimulated SLC, but increased proliferation of Con-A stimulated CLNC; and 42–46, reduced food intake and growth. Suppressed proliferation of Con-A stimulated SLC from CD rats was not alleviated following removal of plastic-adherent lymphoid cells from the SLC suspension. Equivalent proliferation was exhibited by Con-A stimulated SLC from CD rats and NASLC from CD and CA rats. In conclusion, reduced T-lymphocyte proliferation in copper-deficiency: is influenced by the tissue source of lymphoid cells; is not due to impaired ability of T-lymphocytes to proliferate; and may be due to imparied enhancer function for T-lymphocyte proliferation by macrophages.

Isolation of a (copper, zinc)-thionein from the small intestine of neonatal rats.

Abstract A Cu- and Zn-binding protein was isolated from the small intestine of 5-day-old rats whose only source of these metals was maternal milk. Elution behavior on Sephadex G-75 indicated that the molecular weight of this protein was about 9,000 daltons. Furthermore, the protein exhibited low absorbance at 280 nm, was separated into two subfractions by DEAE Sephadex chromatography and had low aromatic amino acid and high cysteine content. The protein, therefore, meets the criteria for classification as a metallothionein. Evidence suggested that the metallothionein is located primarily in the distal portion of the small intestine.

Intracellular calcium mobilization in rat platelets is adversely affected by copper deficiency

The influence of copper deficiency on the mobilization of Ca2+ from intracellular stores following ionomycin treatment or thrombin activation of rat platelets was examined using the fluorescent indicator, fura-2, to measure changes in cytosolic Ca2+ concentration ([Ca2+]i). Platelets, obtained from copper-deficient and control rats and loaded with fura-2, were suspended in medium containing 1 mM EGTA and no added Ca2+. The size of the internal Ca2+ pools in the suspended platelets was estimated from the rise in [Ca2+]i following maximal discharge of stored Ca2+ by treatment with 1 microM ionomycin. Peak [Ca2+]i following ionomycin treatment was lower in platelets from copper-deficient rats compared to control rats (148 +/- 27 nM vs. 188 +/- 17 nM), suggesting that the size of the Ca2+ storage pools was decreased by copper deficiency. Furthermore, once internal Ca2+ stores were discharged by ionomycin, [Ca2+]i remained elevated in platelets from copper-deficient rats, but decreased in control rats. These data indicate that copper deficiency may inhibit the efflux of Ca2+ from platelets after its release from internal stores by ionomycin treatment. In platelets from copper-deficient and control rats, stimulation with 0.1 U/ml thrombin led to rapid rise followed by a slow decay in [Ca2+]i. However, peak [Ca2+]i was lower in platelets from copper-deficient rats than in control rats (94 +/- 19 nM vs. 131 +/- 16 nM). These findings imply that by reducing the amount of Ca2+ available for release from intracellular stores, copper deficiency also reduces [Ca2+]i following thrombin activation in the absence of external Ca2+.

Temporal changes in heart size, hematocrit and erythrocyte membrane protein in copper-deficient rats

Abstract The temporal relationship between Cu depletion and the development of anemia, cardiac hypertrophy and altered erythrocyte membrane protein composition was investigated. Male, weanling Sprague-Dawley rats were fed diets containing either

Dietary supplementation with t-butylhydroquinone reduces cardiac hypertrophy and anemia associated with copper deficiency in rats

Abstract The effect of dietary supplementation with the antioxidant, t-butylhydroquinone (TBHQ), on some of the cardiovascular consequences of copper deficiency was investigated. Rats were fed copper-deficient (CuD) diet containing 0.3 μg Cu/g of diet that were either nonsupplemented or supplemented with TBHQ (supplied in the dietary safflower oil at a concentration of 0.02%). Control rats were fed copper adequate (CuA) diet containing >5.0 μg Cu/g (CuA) that also were either nonsupplemented or supplemented with TBHQ. After five weeks, rats consuming CuD diet supplemented with TBHQ exihibited plasma copper concentrations, ceruloplasmin activities, and liver and heart copper concentrations that were significantly (P

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.