Exercise down-regulates hepatic fatty acid synthase in streptozotocin-treated rats.

An acute bout of prolonged exercise has been shown to decrease hepatic fatty acid synthase (FAS) mRNA and activity induced by high carbohydrate diets. The purpose of the current study was to examine the role of insulin in this exercise down-regulation of FAS. Sixty-four male Wistar rats were randomly divided into normal and streptozotocin (STZ)-treated diabetic groups. After being starved for 48 h and refed a high cornstarch (C) or fructose (F) diet for 10 h, one half of each group of rats was killed after an acute bout of prolonged exercise (E), while the other half of the group was killed in the rested state. STZ treatment suppressed plasma insulin and elevated plasma glucagon levels along with a severe hyperglycemia. FAS mRNA levels decreased by 60% (P < 0.05) with STZ treatment but were 250% higher in F-fed versus C-fed rats. E abolished F-induced FAS mRNA levels in both normal and STZ rats and decreased plasma glucose concentration in STZ rats (P < 0.05). F-fed normal rats showed twofold higher hepatic FAS activity than did C-fed normal rats and this dietary induction was abolished by STZ (P < 0.05). FAS activity in normal rats was not affect by E and was increased with E in STZ rats. Nuclear protein binding to the insulin response sequence was not affected by STZ or diet and increased with E (P < 0.05). Carbohydrate response element binding was greater with F- versus C-feeding (P < 0.05) but unaffected by E. E enhanced inverted CCAAT-box element binding regardless of diet and STZ. We conclude that although insulin status had a great influence on FAS gene expression, E-induced down-regulation of FAS mRNA was not mediated by altered insulin response sequence binding but primarily by increased inverted CCAAT-box element binding to the FAS promoter and/or decreased concentration of carbohydrate metabolites.

Superoxide dismutase gene expression is activated by a single bout of exercise in rat skeletal muscle.

The goal of this experiment was to examine contraction-mediated activation of superoxide dismutase (SOD) gene expression in rat superficial vastus lateralis (SVL, type IIb) and deep vastus lateralis (DVL, type IIa) muscles. Female Sprague-Dawley rats were randomly divided into exercise (E) and control (C) groups that were sacrificed at 0, 1, 2, 4, 10, 24, and 48 h (n=6) following an acute bout of treadmill exercise (25 m/min, 5% grade) to exhaustion (running time approximately equals 1 h). Nuclear factor-kappaB (NF-kappaB) in DVL and SVL showed maximal binding at 2 and 10 h respectively, and remained elevated. Activator protein-1 (AP-1) showed maximal binding at 1 h post-exercise, and returned to resting levels at 10 h in both muscles. Mn SOD mRNA abundance in the DVL was increased at 0 (P<0.01), 1, and 2 h (P<0.05) post-exercise, whereas Mn SOD protein was unchanged. In SVL, Mn SOD mRNA abundance was not altered by exercise, whereas Mn SOD protein content was increased at 10 (P<0.05) and 24 h (P<0.075) post-exercise. CuZn SOD mRNA was unchanged with exercise in DVL and SVL, but CuZn SOD protein was elevated 48 h after exercise in both DVL and SVL (P<0.01). Activities of Mn SOD, CuZn SOD and total SOD showed no change with exercise in either muscle examined. These findings indicate that an acute bout of exercise can increase binding of NF-kappaB and AP-1 in both SVL and DVL, which may stimulate Mn SOD mRNA transcription in the more oxidative type DVL muscle. The increased CuZn SOD protein contents seen post-exercise, without increases in mRNA abundance in both DVL and SVL, suggest a translational mechanism in this SOD isoform.

Superoxide dismutase gene expression in skeletal muscle: fiber-specific adaptation to endurance training.

The effects of endurance training on the enzyme activity, protein content, and mRNA abundance of Mn and CuZn superoxide dismutase (SOD) were studied in various phenotypes of rat skeletal muscle. Female Sprague-Dawley rats were randomly divided into trained (T, n = 8) and untrained (U, n = 8) groups. Training, consisting of treadmill running at 27 m/min and 12% grade for 2 h/day, 5 days/wk for 10 wk, significantly increased citrate synthase activity (P < 0. 01) in the type I (soleus), type IIa (deep vastus lateralis, DVL), and mixed type II (plantaris) muscles but not in type IIb (superficial vastus lateralis, SVL) muscle. Mitochondrial (Mn) SOD activity was elevated by 80% (P < 0.05) with training in DVL. SVL and plantaris muscle in T rats showed 54 and 42% higher pooled immunoreactive Mn SOD protein content, respectively, than those in U rats. However, no change in Mn SOD mRNA level was found in any of the muscles. CuZn SOD activity, protein content, and mRNA level in general were not affected by training, except for a 160% increase in pooled CuZn SOD protein in SVL. Training also significantly increased glutathione peroxidase and catalase activities (P < 0.05), but only in DVL muscle. These data indicate that training adaptations of Mn SOD and other antioxidant enzymes occur primarily in type IIa fibers, probably as a result of enhanced free radical generation and modest antioxidant capacity. Differential training responses of mRNA, enzyme protein, and activity suggest that separate cellular signals may control pre- and posttranslational regulation of SOD.

Exercise attenuates nuclear protein binding to gene regulatory sequences of hepatic fatty acid synthase.

The effect of an acute bout of exhaustive exercise on hepatic fatty acid synthase (FAS) gene expression was examined in rats. Female Sprague-Dawley rats (age 8 wk) were fasted for 48 h (F, n = 6), or fasted, refed a high-fructose diet for 6 h, and killed at rest (R, n = 6) or killed after running on a treadmill at 27 m/min and 5% grade for 88 +/- 7 min (E, n = 6). Gel mobility shift assay indicated that R rats had twofold higher liver nuclear protein binding to oligonucleotides corresponding to the insulin responsive sequence (-71/-50) and carbohydrate response element (+283/+303) on the FAS promoter, compared with F rats. Exercise severely attenuated this binding in liver nuclear extracts to the levels seen in F rats. Competition and supershift experiments revealed that the bound protein complexes contained the upstream stimulatory factors. Nuclear run-on experiment revealed a 49-fold increase in transcription rate of the FAS gene in R vs. F rats, whereas exercise suppressed the transcription rate. FAS mRNA abundance and FAS enzyme activity were dramatically increased with refeeding but were unaltered by exercise. The results reveal that dietary induction of hepatic FAS is stimulated by increased nuclear protein binding to insulin responsive sequence and carbohydrate response element, whereas exhaustive exercise attenuates the binding, which may precede downregulation of FAS mRNA and enzyme synthesis reported in our previous work (M. A. Griffiths, R. Fiebig, M. T. Gore, D. H. Baker, K. Esser, L. Oscai, and L. L. Ji. J. Nutr. 126, 1959-1971, 1996).

Free radical activity, antioxidant enzyme, and glutathione changes with muscle stretch injury in rabbits.

The present study investigated changes in rate of free radical production, antioxidant enzyme activity, and glutathione status immediately after and 24 h after acute muscle stretch injury in 18 male New Zealand White rabbits. There was no change in free radical production in injured muscles, compared with noninjured controls, immediately after injury (time 0; P = 0.782). However, at 24 h postinjury, there was a 25% increase in free radical production in the injured muscles. Overall, there was an interaction (time and treatment) effect (P = 0.005) for free radical production. Antioxidant enzyme activity demonstrated a treatment (injured vs. control) and interaction effect for both glutathione peroxidase (P = 0.015) and glutathione reductase (P = 0.041). There was no evidence of lipid peroxidation damage, as measured by muscle malondialdehyde content. An interaction effect occurred for both reduced glutathione (P = 0.008) and total glutathione (P = 0.015). Morphological analysis (hematoxylin and eosin staining) showed significant polymorphonuclear cell infiltration of the damaged region at 24 h postinjury. We conclude that acute mechanical muscle stretch injury results in increased free radical production within 24 h after injury. Antioxidant enzyme and glutathione systems also appear to be affected during this early postinjury period.

Exercise training down-regulates hepatic lipogenic enzymes in meal-fed rats: fructose versus complex-carbohydrate diets.

The maximal activity and mRNA abundance of hepatic fatty acid synthase (FAS) and other lipogenic enzymes were investigated in rats meal-fed either a high fructose (F) or a high cornstarch (C) diet. The diet contained 50% F or C (g/100 g), casein (20%), cornstarch (16.13%), corn oil (5%), minerals (5.37%), vitamins (1%) and Solka-floc (2%). Female Sprague-Dawley rats (n = 44) were randomly divided into C or F groups that were meal-fed for 3 h/d; each group was subdivided into exercise-trained (T) and untrained (U) groups. Treadmill training was performed 4 h after the initiation of the meal at 25 m/min, 10% grade for 2 h/d, 5 d/wk, for 10 wk. Rats were killed 9 h after the meal and 27 h after the last training session. F-fed rats had significantly higher activities of all lipogenic enzymes assayed and mRNA abundance of FAS and acetyl-coenzyme A carboxylase (ACC) than C rats (P < 0.05). Concentrations of plasma insulin and glucose and liver pyruvate were not altered by F feeding. Proportions of the fatty acids 18:2 and 20:4 were lower, whereas those of 16:0 and 16:1 were higher, in livers of F than of C rats (P < 0.05). Training decreased FAS activity by 50% (P < 0.05), without affecting FAS mRNA level in C rats; this down-regulation was absent in the F rats. ACC mRNA abundance tended to be lower in CT than in CU rats (P < 0.075). L-Type pyruvate kinase activity was lower in FT than in FU rats (P < 0.05), whereas other lipogenic enzyme activities did not differ between T and U rats of each diet group. We conclude that hepatic lipogenic enzyme induction by high carbohydrate meal feeding may be inhibited by exercise training and that a fructose-rich diet may attenuate this training-induced down-regulation.

Spaceflight downregulates antioxidant defense systems in rat liver.

Liver antioxidant enzyme activities, mRNA abundance, and glutathione (GSH) status were investigated in male Sprague-Dawley rats placed in an enclosure module aboard Space Shuttle STS-63 for 8 d (F, n = 6). F animals were compared to rats housed in an enclosure module on the ground (G, n = 9), which simulated the vibration and temperature conditions associated with launch and flight, and rats kept under conventional ground vivarium conditions in individual cages (V, n = 6). Spaceflight significantly decreased catalase, GSH reductase, and GSH sulfur-transferase activities in the liver (p < .05). Neither enzyme activity nor enzyme protein content of Cu-Zn and Mn superoxide dismutase (SOD) was affected by flight. The relative abundance of mRNA for Cu-Zn SOD and catalase was significantly decreased comparing F with G rats (p < .05). Spaceflight resulted in a dramatic decrease of liver GSH, glutathione disulfide, and total GSH contents (p < .01), which were accompanied by a lower gamma-glutamyl transpeptidase activity (p < .05). F rats showed a 47% (p < .05) increase in liver malondialdehyde concentration compared to G and V rats. Liver protein content was not affected by flight. These results indicate that spaceflight can downregulate antioxidant defense capacity and elicit an oxidative stress in the liver.

Oxidative stress and aging. Role of exercise and its influences on antioxidant systems.

Strenuous exercise is characterized by an increased oxygen consumption and disturbance of intracellular prooxidant-antioxidant homeostasis. At least three biochemical pathways, that is, mitochondrial electron transport chain, xanthine oxidase, and polymorphoneutrophil have been identified as potential sources of intracellular free radical generation during exercise. These deleterious reactive oxygen species pose a serious threat to the cellular antioxidant defense system, such as diminished reserve of antioxidant vitamins and glutathione, and have been shown to cause oxidative damage in exercising and/or exercised muscle and other tissues. However, enzymatic and nonenzymatic antioxidants have demonstrated great versatility and adaptability in response to acute and chronic exercise. The delicate balance between prooxidants and antioxidants during exercise may be altered with aging. Study of the complicated interaction between aging and exercise under the influence of reactive oxygen species would provide more definitive information as to how much aged individuals should be involved in physical activity and whether supplementation of nutritional antioxidants would be desirable.

Endurance training alters antioxidant enzyme gene expression in rat skeletal muscle.

The effects of endurance training on gene expression of superoxide dismutase (SOD) and glutathione peroxidase (GPX) were investigated in type 2a and 2b skeletal muscles, as well as heart and liver, in the rat. Female Sprague-Dawley rats (4 months old, 300-320 g) were randomly divided into a trained (T, n = 11) and a control (C, n = 10) group and were pair fed a diet consisting of 66% cornstarch and 34% basal diet that contained all essential nutrients. Training was conducted on a treadmill at 25 m x min(-1), 10% grade for 2 h per day, 5 days per week for 10 weeks, resulting in a 79% (p < 0.01) increase in citrate synthase activity in the deep portion of vastus lateralis muscle (DVL, type 2a). Cu-Zn SOD activity was 35% higher (p < 0.01) in DVL of T versus C rats, and Cu-Zn SOD mRNA abundance showed a 125% increase with training (p < 0.05). Cu-Zn SOD protein content was not altered in DVL, but increased significantly (p < 0.05) in the superficial portion of vastus lateralis (type 2b) with training. Trained rats showed a 66% higher (p < 0.05) Mn SOD protein content in DVL, but Mn SOD activity and mRNA abundance were not affected. Training also significantly increased GPX activity by 62% (p < 0.05), without changing its mRNA abundance, in the DVL. Heart and liver showed a 112 and 58% increase (p < 0.01) in Cu-Zn SOD mRNA abundance with training, respectively, but no other training adaptation was detected. These data indicate that endurance training can promote gene expression of muscle antioxidant enzymes in a fiber-specific manner. Training appears to upregulate Cu-Zn SOD mRNA abundance in a number of aerobic tissues, whereas Mn SOD and GPX induction observed in DVL may occur at the post-transcriptional levels.

Rigorous swim training impairs mitochondrial function in post-ischaemic rat heart.

The effect of rigorous swim training (6 h day-1, 5 days week-1 for an average of 191 h) on mitochondrial respiratory function was investigated in rat heart subjected to in vivo ischaemia reperfusion (I-R). Mitochondria was isolated from the risk region of the left ventricle subjected to 60 min occlusion of the main left coronary artery followed by 30 min reperfusion. Heart weight and heart-to-body weight ratio was increased by 21 and 28% (P < 0.01), respectively, in the trained (T, n = 15) vs. control rats (C, n = 20). I-R per se showed minimal effect on heart mitochondria regardless of training status. In sham, state 4 respiration rate was 26 and 32% (P < 0.05) lower in T vs. C rats, using malate-pyruvate (M-P) and 2-oxoglutarate (OG) as substrates, respectively. Training also reduced state 3 respiration by 28% (M-P) and 50% (OG) (P < 0.01). The respiratory control index (RCI) was unaltered in T with M-P, but decreased with OG (P < 0.01). In vitro exposure to superoxide radicals severely reduced state 4 and 3 respiration and RCI, but T hearts showed greater reductions of state 4 and 3 rates than C. Mitochondria from T hearts also revealed a greater state 4 inhibition by H2O2 and HO. compared with C. A lower glutathione content and a higher gamma-glutamyl transpeptidase activity (P < 0.05) was observed in T vs. C. It is concluded that rigorous swim training impairs heart mitochondrial function, making them more susceptible to in vivo and in vitro oxidative stress, and that this damaging effect may be related to a diminished glutathione reserve.

Exercise down-regulates hepatic lipogenic enzymes in food-deprived and refed rats.

The effects of an acute bout of prolonged exercise on the activities of several hepatic lipogenic enzymes and the abundance of fatty acid synthase (FAS) mRNA were evaluated using a food deprivation-refeeding protocol in which diets contained 50% of the energy from either fructose or cornstarch. Food was withheld from male rats for 48 h and refed for 0, 4, 8, 12, 24 or 48 h. At each time point, half of each dietary group was subjected to a single bout of treadmill running until exhaustion and killed immediately. The other half of each group rested without food for the same amount of time before being killed. Exercise significantly decreased FAS activity by 57, 46, 10, 26 and 70% at 4, 8, 12, 24 and 48 h of refeeding, respectively, in the fructose-fed rats; and by 70 and 63% at 24 and 48 h of refeeding, respectively, in the cornstarch-fed rats. Activities of L-type pyruvate kinase and glucose 6-phosphate dehydrogenase were significantly decreased after exercise in the fructose-fed, but not cornstarch-fed rats. In rested rats, FAS mRNA abundance increased approximately fourfold above the unfed levels after 8 and 12 h of refeeding. Exercise attenuated the diet-induced increases in FAS mRNA abundance. At 8 h of refeeding, both cornstarch- and fructose-fed exercised rats had 71% (P < 0.05) of the FAS mRNA levels of their rested counterparts; at 12 h, these exercised rats showed only 46 and 27% (P < 0.05) of FAS mRNA levels compared with rested rats fed the same diet. We conclude that dietary induction of FAS activity and mRNA abundance can be inhibited by prolonged exercise, suggesting that exercise may influence FAS transcription and/or mRNA stability.

Effect of acute exercise on glutathione deficient heart.

The role of glutathione (GSH) in myocardial antioxidant defense was investigated in Swiss-Webster mice either performing swim exercise to exhaustion or rested in both the GSH adequate (GSH-A) and GSH deficient (GSH-D) states. GSH deficiency was accomplished by injecting mice with L-buthionine [S,R]sulfoximine (BSO; 2 nmol/kg body wt, i.p.) and providing BSO (20 mM) in drinking water for 12 days. GSH and glutathione disulfide (GSSG) contents in the GSH-D hearts were decreased to 10 and 8%, respectively, of those in the GSH-A mice. This decrease was associated with a significant decline of the total glutathione level in the liver, skeletal muscle and plasma. Myocardial GSH peroxidase and GSH sulfur-transferase activities decreased significantly following GSH deficiency, whereas superoxide dismutase activity was significantly elevated. GSH deficiency did not affect exercise endurance performance. However, exhaustive exercise decreased GSH content in the myocardium of the GSH-A and GSH-D mice by 22 and 44% (p < 0.05), respectively. The GSH:GSSG ratio was not altered significantly following exercise because of a concomitant decrease in GSSG (p < 0.05). gamma-Glutamyltranspeptidase activity was significantly increased after exercise, especially in the GSH-D hearts (72%; p < 0.05). GSH content after exercise correlated negatively with exercise time in both GSH-A and GSH-D mice (p < 0.05). These data indicate that GSH is actively used in the myocardium during prolonged exercise at moderate intensity and that GSH deficiency is tolerated by the heart, possibly compensated for by an increased GSH uptake from the plasma.

Aging and exercise training in skeletal muscle: responses of glutathione and antioxidant enzyme systems.

Glutathione (GSH) content and antioxidant enzyme activities were investigated in skeletal muscle of young, adult, and old male Fischer 344 rats. Furthermore, the effect of 10 wk of exercise training on these antioxidant systems was evaluated at all ages. In the soleus muscle, GSH concentration increased markedly with age, with no significant change in glutathione disulfide (GSSG) content. Training caused a 30% decrease of GSH (P < 0.05) in the soleus of young rats and a reduction of the GSH-to-GSSG ratio at all ages. Activity of gamma-glutamyl transpeptidase (GGT), a key enzyme for GSH uptake by muscle, was also significantly decreased with training. GSH, GSSG, and the GSH-to-GSSG ratio were not altered with aging or training in the deep portion of vastus lateralis muscle (DVL). Activities of GSH peroxidase (GPX), GSSG reductase (GR), superoxide dismutase (SOD), catalase (CAT), and GSH sulfur-transferase were increased significantly with aging in both soleus and DVL. In DVL, training increased GPX and SOD activities in the young rats, whereas in soleus, training decreased GR and CAT activities in the adult rats and GGT and CAT activities in the old rats. Muscle lipid peroxidation was significantly increased with aging in both DVL and soleus but was not affected by training. These data indicate that aging may cause not only an overall elevation of antioxidant enzyme activities but also a fiber-specific adaptation of GSH system in skeletal muscle. Exercise training, although increasing selective antioxidant enzymes in the young rats, does not offer additional protection against oxidative stress in the senescent muscle.

Lysis of left ventricular thrombi with urokinase.

In 16 patients with recent myocardial infarction (3 to 12 week old) and with large left ventricular thrombi systemic thrombolysis with urokinase was performed. Left ventricular thrombi were diagnosed by two-dimensional echocardiography; in all patients the mural thrombus was located in the area of recent myocardial infarction. Each of three patients suffered an embolic episode before the initiation of thrombolytic therapy and the episode caused a stroke in one. Urokinase was infused intravenously at a rate of 60,000 U/hr for 2 to 8 days in combination with intravenous heparin (200 units/kg X 12 hr). Left ventricular thrombi were successfully lysed in 10 of 16 patients, as determined by two-dimensional echocardiography. In four of the six remaining patients only partial thrombolysis was achieved and in two thrombolytic treatment failed. There was no evidence of embolic events during thrombolysis in any of the 16 patients. The success of thrombolysis seemed to depend on the age of the thrombus: the thrombus was dissolved in eight of nine patients undergoing thrombolysis within 4 weeks of the acute myocardial infarction vs in two of seven patients receiving treatment later (p = .057). The presence of a left ventricular aneurysm or depressed left ventricular function also appeared to reduce the likelihood of successful thrombolysis. All patients were discharged on oral anticoagulants. At 6 months follow-up (n = 9) no recurrence of left ventricular thrombus was found. These results show that left ventricular thrombi can be safely lysed by intravenous urokinase. However, for better definition of the risk and benefit of this new therapy further investigation is necessary.

The effect of corticosterone on the fluxes of 3H-normetanephrine into and out of the extraneuronal compartments of the perfused rat heart.

1. Hearts were obtained from pargyline-pretreated rats and perfused with 40 nM 3H-normethanephrine for 21 min. 87 muM corticosterone reduced the accumulation of 3H-normethanephrine in the heart. 2. When the initial perfusion with 3H-normetanephrine was followed by perfusion of the hearts with amine-free solution for 90 min, efflux of radioactivity was determined. Analysis of efflux curves showed that, apart from its distribution into extracellular space and fluid content of cardiac cavities, radioactivity distributed mainly into one corticosterone-sensitive, extra-neuronal compartment (compartment III, half time for efflux about 6 min). 3. When corticosterone was added to the wash out solution only, it failed to affect the efflux of radioactivity. 4. Although monoamine oxidase was inhibited, some deaminated metabolites of normetanephrine were detected in the efflux. Efflux of 3H-metabolites was monophasic in about half the hearts (short half time corresponding to that of compartment III) and biphasic in the other half. The early phase of efflux (short half time) seemed to represent efflux of newly formed metabolite(s), while the late phase (if present) appeared to be due to efflux of metabolites formed early in the experiment. 5. While corticosterone is known to inhibit the extraneuronal influx and efflux of catecholamines, it appears to impair the influx of normetanephrine without affecting the efflux.