Frederick W. Stratman

Antioxidant enzyme systems in rat liver and skeletal muscle: Influences of selenium deficiency, chronic training, and acute exercise☆

The influences of selenium deficiency (Se-D), chronic training, and an acute bout of exercise on hepatic and skeletal muscle antioxidant enzymes, i.e., superoxide dismutase (SOD), catalase, and glutathione peroxidase (GPX), as well as glutathione S-transferase (GST) and tissue lipid peroxidation, were investigated in post-weaning male Sprague-Dawley rats. Se-D per se depleted GPX in both liver and skeletal muscle but had no effect on SOD or catalase activity. One hour of treadmill running (20 m/min, 0% grade and 27 m/min, 15% grade for untrained and trained rats, respectively) significantly elevated hepatic catalase and cytosolic SOD activity; more prominent activations were found in the Se-D or untrained rats, whereas skeletal muscle antioxidant enzymes were little affected. Ten weeks of training (1 h/day, 5 days/week at 27 m/min, 15% grade) increased hepatic mitochondrial SOD by 23% (P less than 0.05) in Se-D rats. Both hepatic mitochondrial and cytosolic GPX were decreased by training whereas GPX was increased twofold in skeletal muscle mitochondria. Se-independent GPX was elevated by training only in the skeletal muscle mitochondria of Se-D rats. Lipid peroxidation (malondialdehyde formation) was increased by an acute bout of exercise in hepatic mitochondria of the untrained rats and in skeletal muscle mitochondria of the Se-D rats. These data indicate that antioxidant enzymes in liver and skeletal muscle are capable of adapting to selenium deficiency and exercise to minimize oxidative injury caused by free radicals.

The isolation of hormone-sensitive rat hepatocytes by a modified enzymatic technique

Abstract Hepatocytes that are similar to the perfused liver in glucagon sensitivity can be obtained in a high, reproducible yield by modifications of the well-known enzymatic technique for the preparation of isolated liver cells. The major modifications are: (a) a simple, economic, and temperature-controlled apparatus for the recirculating perfusion of the isolated rat liver; (b) the use of substrate-fortified calcium-free Krebs-Henseleit bicarbonate buffer; and (c) high perfusion rates, which lead to the isolation of hepatocytes with normal ultrastructure and metabolic activities. From 4 × 10 8 to 5 × 10 8 cells can be routinely isolated from an 8- to 10-g liver independent of the collagenase preparations applied. The rat liver cells are viable (90–95%) by various criteria including electron microscopy and exclusion of 0.2% trypan blue. When studying various incubation techniques, it was observed that the use of gelatin in the medium is preferred as compared to albumin Fraction V or fatty acid-free albumin which tended to inhibit gluconeogenic rates from various substrates in calcium-free medium. Addition of calcium chloride to the incubation medium strikingly improved gluconeogenesis from lactate. Various procedures for calculating the number of cells corresponding to 1 g wet liver tissue are discussed in detail.

The influence of ammonium and calcium ions on gluconeogenesis in isolated rat hepatocytes and their response to glucagon and epinephrine

Abstract The use of n-butylmalonate as an inhibitor of malate transport from mitochondria and of aminooxyacetate as an inhibitor of glutamate-aspartate transaminase indicated that rat liver hepatocytes employ the aspartate shuttle for gluconeogenesis from lactate which supplies reducing equivalents to the cytosolic NAD system. In contrast, malate is transported from mitochondria to cytosol for gluconeogenesis from pyruvate. This conclusion is corroborated by the finding that the addition of ammonium ions enhances gluconeogenesis from lactate but inhibits glucose formation from pyruvate. In hepatocytes, glucagon and epinephrine have relatively little effect on glucose synthesis from lactate. Ammonium ions permit both of these hormones to exert their usual stimulation of gluconeogenesis from lactate. Calcium ions (1.3 m m ) enhance gluconeogenesis from lactate and from lactatepyruvate mixtures (10:1). The stimulatory effects of Ca2+ and NH4+ are additive and, when lactate is the substrate, the rates of gluconeogenesis achieved are so high as to preclude further stimulation by glucagon.

Antioxidant enzyme response to selenium deficiency in rat myocardium

Influences of dietary selenium (Se) deficiency, physical training and an acute bout of exercise on myocardial antioxidant enzyme activity, lipid peroxidation and related biochemical properties were investigated in post-weanling male Sprague-Dawley rats. An experimental group was fed a diet containing less than 0.01 mg Se/kg and had free access to distilled water (Se-D), whereas control rats were supplemented with 0.5 mg Se/l in drinking water (Se-A). Se deficiency depleted heart mitochondrial and cytosolic Se-dependent glutathione peroxidase activity to 24 and 3%, respectively, of those in Se-A rats. Heart mitochondrial superoxide dismutase (Mn SOD) activity was 24% higher (p less than 0.05) in Se-D than in Se-A rats. Cytosolic (copper-zinc) SOD and catalase activities were not altered, whereas glutathione S-transferase activity was significantly decreased in Se-D (p less than 0.01). Myocardial antioxidant enzyme activities were not affected by either training or an acute exercise bout. Heart lipid peroxidation and activities of several enzymes in substrate metabolism were also unaffected by Se or exercise. It is concluded that rat heart has sufficient reserve of antioxidant enzyme capacity in coping with oxidative stress imposed by Se deficiency or exercise. The adaptation of Mn SOD may reveal its potential role in myocardial antioxidant defense.

Amino Acid Metabolism During Exercise in Trained Rats: The Potential Role of Carnitine in the Metabolic Fate of Branched-Chain Amino Acids

The influence of endurance training and an acute bout of exercise on plasma concentrations of free amino acids and the intermediates of branched-chain amino acid (BCAA) metabolism were investigated in the rat. Training did not affect the plasma amino acid levels in the resting state. Plasma concentrations of alanine (Ala), aspartic acid (Asp), asparagine (Asn), arginine (Arg), histidine (His), isoleucine (Ile), leucine (Leu), lysine (Lys), methionine (Met), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), and valine (Val) were significantly lower, whereas glutamate (Glu), glycine (Gly), ornithine (Orn), tryptophan (Trp), tyrosine (Tyr), creatinine, urea, and ammonia levels were unchanged, after one hour of treadmill running in the trained rats. Plasma concentration of glutamine (Glu), the branched-chain keto acids (BCKA) and short-chain acyl carnitines were elevated with exercise. Ratios of plasma BCAA/BCKA were dramatically lowered by exercise in the trained rats. A decrease in plasma-free carnitine levels was also observed. These data suggest that amino acid metabolism is enhanced by exercise even in the trained state. BCAA may only be partially metabolized within muscle and some of their carbon skeletons are released into the circulation in forms of BCKA and short-chain acyl carnitines.

Total cholesterol and HDL-cholesterol changes during acute, moderate-intensity exercise in men and women.

Chronic endurance exercise training has been associated with decreased levels of total cholesterol and increased HDL-cholesterol. To our knowledge rapid changes in cholesterol and HDL-cholesterol during acute exercise have not been described under controlled conditions. We studied 28 subjects (14 males and 14 females) during bicycle exercise for 40 min at a work intensity of 55% of their maximal oxygen consumption. Total and HDL-cholesterol levels were measured (and LDL-cholesterol calculated) at rest, 10, 20, 30, and 40 min of exercise, and 15 min postexercise. There was a significant (p less than 0.001) increase in HDL-cholesterol levels at 10 min of exercise (58.8 +/- 13.9 mg/dl, mean +/- SD) above rest (53.1 +/- 13.4 mg/dl) for all subjects. This increase persisted (p less than 0.001) at all time points throughout the exercise session, but declined by 15 min postexercise. There was a small, insignificant decline in LDL-cholesterol. It is concluded that apparent favorable changes in lipoprotein patterns occur acutely, and are sustained during short-term, moderate intensity exercise. Analyses of these changes appears necessary if the biochemical mechanisms which underlie these metabolic alterations are to be elucidated.

Dehydroepiandrosterone and a β-agonist, energy transducers, alter antioxidant enzyme systems: Influence of chronic training and acute exercise in rats☆

We examined the influence of dehydroepiandrosterone (DHEA), a beta-agonist, and exercise training on enzymes that detoxify toxic oxygen species. Feeding 0.4% DHEA decreased hepatic cytosolic (c) selenium-dependent glutathione peroxidase (GPX), (-26%, P less than 0.0001) and increased hepatic mitochondrial (m) Mn superoxide dismutase (SOD), (+38%, P less than 0.001). DHEA decreased myocardial c-GPX (-21%, P less than 0.05) when compared to a beta-agonist (beta A; L644969 Merck and Co.) fed at 5 ppm but neither differed from the Control (C). In contrast, the beta A increased hepatic m-GPX (+25%, P less than 0.05). In skeletal muscle, DHEA and beta A decreased muscle c-GPX by 20 and 12%, respectively (P less than 0.0009). DHEA increased both muscle (+20%, P less than 0.01) and myocardial (+20%, P less than 0.05) c-glutathione S-transferase (GST) over beta A (+20%, P less than 0.01) but neither was significantly different from C. Similar to DHEA, chronic training (Tr) (1 h/day, 5 days/week at 27 m/min, 15% grade on treadmill) decreased hepatic c-GPX (-16%, P less than 0.003). Tr elevates muscle c-GPX (+36%, P less than 0.05) in C. Tr increased myocardial c-GPX by 28% in the beta A-treated rats, whereas Tr decreased myocardial c-GPX by 22% in the C (P less than 0.05, interaction). One hour of acute exercise (Ex) (70% VO2 max relative work load) decreased hepatic homogenate catalase (-12%, P less than 0.02) and increased hepatic m-Mn SOD (+28%, P less than 0.03). Ex decreased myocardial c-GST (P less than 0.05) only in the DHEA-treated rats. DHEA and Tr may improve efficiency of oxygen utilization at the tissue level with lower antioxidant enzyme activity in liver and locally protective up-regulation in muscle. beta A stresses oxygen utilization systems and liver responds by up-regulation of antioxidant enzymes. The increase in myocardial c-GPX activity in the beta A-treated group may be a protective effect against indirect catecholamine-induced myocardial necrosis which results from free radical generation.

Pyruvate uptake in rat liver mitochondria: Transport or adsorption?

Studies were performed in our laboratory to investigate certain aspects of pyruvate translocation into rat liver mitochondria, which is still a matter of controversy. Even though evidence has been collected in recent years showing that monocarboxylate uptake in mitochondria is not a controlled and carrier-limited translocation [ 1, 21, Papa et al. [3] reported recently that pyruvate is transported through a specific translocator into mitochondria. The method selected for our study was the technique of rapid centrifugation (with or without silicone layer), which was established as the method of choice for many carboxylic acid [l] and adenine nucleotide [ 1,4] transport and exchange studies. Mitochondrial terminology such as “uptake” and “transport” (translocation of a substrate from one side of the membrane to the other) usually does not distinguish precisely between a translocation through a membrane system or a simple “surface limited” or “bulk phase limited” [5] adsorption of anions or cations on proteins and lipids. For these transport studies, we prepared different denatured, metabolically and structurally destroyed mitochondria as a control to normal mitochondria. We present evidence that it is not possible to justify the kinetic treatment [3] of the data obtained with the method of rapid centrifugation as representing active transport. Adsorption of pyruvate to mitochondrial proteins and/or lipids rather than specific transport to the matrix space appears to account for the binding of pyruvate by rat liver mitochondria.

Increased acetyl carnitine in rat skeletal muscle as a result of high-intensity short-duration exercise: Implications in the control of pyruvate dehydrogenase activity

Several functions have been proposed for carnitine @-hydroxy-y-trimethylammonium butyrate) such as: (i) The oxidation of fatty acids in tissues by serving as a carrier of ‘activated’ fatty acids across the mitochondrial inner membrane to the site of /3-oxidation [ 11; (ii) A ‘buffer’ for acetyl-coenzyme A in spermatozoa by forming acetyl carnitine and coenzyme A, via acetyl-CoA:carnitine O-acetyl-transferase (EC 2.3.1.7) (CAT) [2]; (iii) A factor in branched chain amino acid oxidation in skeletal muscle for removal as branched chain acyl-CoA esters out of mitochondria to other tissues for further oxidation [3]. The high activity of carnitine transacetylase in mitochondria from various tissues is highly correlated with the necessity of carnitine esters for fatty acid oxidation [4]. This enzyme is absent in both the bee and fly species which utilize only carbohydrates and highly active in the locust which utilizes fatty acids during flight [5,6]. However, in the blowfly flight muscle, which is rich in carnitine but deficient in fatty acid oxidase, carnitine is used in the metabolism of pyruvate especially during transition from rest to rapid contraction [ 71. Exercise increases pyruvate generation by stimulating glucose transport into the mammalian muscle cell as well as by enhancing glycogenolysis and glycolysis,

Artifacts in protein synthesis by mitochondria in vitro.

Protein synthesis by mitochondria in viva and irl vitro has been the subject of an increasing number of publications in recent years [ 1,2]. The importance of the mechanism of mitochondrial biogenesis shifted most investigations from the more complex ill vivo studies to isolated in vitro systems. One of the major disadvantages of using isolated mitochondria is the very low incorporation rate of amino acids into the few mitochondria proteins which can be coded for by mitochondrial DNA. As we will show in this paper, there is striking evidence that despite an apparent regulated protein synthesis (temperature-, concentration-, and time-dependency) the “incorporation” of certain amino acids into mitochondrial proteins in vitro reflects rather specific binding to, or chemical interactions with, the mitochondrial protein-lipid structures. Our amino acid “incorporation” rates are quantitatively comparable to others published previously from various laboratories as reflecting true mitochondrial protein synthesis. The chemical basis of the described phenomena will be discussed.