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.

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.

Effects of acute exercise on hepatic lipogenic enzymes in fasted and refed rats.

The effects of an acute bout of prolonged exhaustive exercise on the activities of hepatic lipogenic enzymes have been investigated. Male Sprague-Dawley rats were randomly divided into three groups: fasted for 48 h without refeeding (FA) and fasted for 48 h and refed a diet high in fructose (RF) or in cornstarch (RC). One-half of each group of rats exercised on a treadmill at 20 m/min, 5% grade, until exhaustion and the other half rested for the same amount of time without food. Dietary intakes during refeeding were kept equal between the exercised and rested control animals. Activities of all hepatic lipogenic enzymes measured, i.e., fatty acid synthase (FAS), L-type pyruvate kinase (L-PK), ATP citrate lyase, malic enzyme, and glucose-6-phosphate dehydrogenase, were induced dramatically by fasting-refeeding and were significantly higher in the RF than in the RC rats (P < 0.05). FAS activity was increased 19- and 39-fold, respectively, in the RC and RF rats compared with the FA rats. Exercise decreased FAS activity to approximately one-third of the resting control value in both RC and RF rats (P < 0.05) but not in FA rats. L-PK activity was elevated by 55% in RC and 100% in RF rats compared with FA rats (P < 0.05). FA and RF rats also showed a reduction of L-PK activity with exercise. No significant alteration of other lipogenic enzymes was observed after exercise.(ABSTRACT TRUNCATED AT 250 WORDS)

Exercise training has a heparin-like effect on lipoprotein lipase activity in muscle.

Lipoprotein lipase (LPL) is anchored with high affinity to heparan sulphate proteoglycans on the luminal surface of the capillary endothelium. The levels of pre-heparin perfusate LPL activity increased from 16 +/- 1 to 145 +/- 6 U/hindlimb (nine-fold increase) in hindlimb muscle of exercise-trained rats measured immediately after the last bout of work. At the same time, post-heparin perfusate LPL activity decreased from 63 +/- 2 to 13 +/- 1 U/hindlimb (p less than 0.001). These results provide evidence that exercise-training has a heparin-like effect on capillary-bound LPL. The total amount of LPL (i.e., pre-heparin perfusate plus post-heparin perfusate) was twofold greater in the hindlimb of the trained animals versus the controls. The effect of exercise on muscle LPL activity appears to last for as long as 5 days after cessation of exercise. Serum triglycerides were reduced 38% and plasma free fatty acids increased fourfold. These results provide evidence that training increases the capacity to remove triglycerides from circulation.

Body fat accretion: a rat model.

A rat model for studying causes of body fat accretion was developed. Employing this model, it was possible to produce a 108 g difference in carcass fat content between two groups of rats simply by selecting the seven fattest and the seven leanest animals from a group of 100. The results of this study provide evidence that the gastrointestinal tract was not a site of caloric wastage, that total energy excreted in feces and urine was directly proportional to caloric intake, that differences in brown adipose tissue thermogenesis were not responsible for the twofold difference in adiposity between the lean and fat rats, and that a family of hepatic lipogenic enzymes was not up-regulated even though the habitual consumption of a diet rich in carbohydrate was greater in the fat vs lean rats. The fat rats also had a greater lean body mass. Therefore, the additional calories consumed by the fat rats could have contributed to their greater adiposity and/or could have been channeled to sustain more active tissue. Finally, evidence was presented to show that the experimental selection process is an important consideration when determining the relationship between caloric intake and body fat content.

Lipase regulation of muscle triglyceride hydrolysis.

The cellular control of intramuscular triglyceride (TG) metabolism involves two major identified lipases: hormone-sensitive lipase (HSL) and lipoprotein lipase (LPL). Recently, the presence of HSL in muscle has been unequivocally demonstrated. However, although it is thought that HSL is responsible for intramuscular TG lipolysis, direct evidence for this is lacking. There is evidence to suggest that HSL and LPL are simultaneously activated under a variety of conditions. The two muscle lipases appear to be turned on by the same signal and function as a coordinated unit in meeting the energy demands of muscle. At a time when HSL is presumably hydrolyzing endogenous TG, LPL is sent to the capillary beds in search of substrate. TG uptake from circulation is highly related to muscle LPL activity. Exercise training increases LPL activity in plasma and in parenchymal cells in muscle. These results suggest that training may increase the capacity to clear TG from circulation and that LPL might have a role in replenishing muscle TG stores that have been decreased with exercise.

Dibutyryl cAMP-induced increases in triacylglycerol lipase activity in developing L8 myotube cultures.

Triacylglycerol (TG) lipase activity, with an alkaline pH optimum, has been identified in the cellular fraction of L8 myotube cultures. This TG lipase activity was stimulated by serum and inhibited by NaCl and protamine sulfate. These characteristics have been classically described for lipoprotein lipase. It was possible to increase the activity of this TG lipase three- to five-fold by incubating the cells with dibutyryl cAMP. Maximal enzyme activity was observed 16 h following the addition of 10-100 microM dibutyryl cAMP to the cultured cells. Enzyme activity returned to control levels 24 h after removal of the nucleotide from the culture medium. Serum-sensitive alkaline TG lipase activity was also identified in five other myotube preparations of cultured muscle cells. The highest levels of activity were found in rat skeletal muscle primary, H9, and L6 cell types. The finding that dibutyryl cAMP is an effective inducer of alkaline TG lipase activity provides us with a valuable model to investigate mechanisms regulating synthesis, compartmentalization, and transport of lipoprotein lipase in muscle.

Hepatic lipid metabolism in exercise and training.

The liver plays a central role in the metabolism of fat. The available data, though sometimes controversial, clearly indicate that muscular exercise affects almost every aspect of fat metabolism in this organ. Neither acute exercise nor training affects total lipid, phospholipid, or cholesterol concentrations in the liver of rats fed chow or low fat diets. However, exercise training reduces accumulation of total hepatic fat and cholesterol in rats fed a fat-rich diet. In addition, training seems to increase both the synthesis and catabolism of cholesterol in the liver in rats fed a chow diet. Production of ketones by the liver increases both during prolonged exercise and during recovery from exercise. Acute prolonged exercise reduces the activities of the enzymes involved in the synthesis of fatty acids and increases oxidation of fatty acids by the liver. This type of work also increases the esterification of fatty acids with the subsequent accumulation of triacylglycerols in this organ. Training does not affect triacylglycerol concentration in the liver of rats fed a chow diet but attenuates its accumulation after a fat-rich diet. Training reduces the postheparin plasma hepatic lipase activity. Finally, it reduces production of triacylglycerols and increases production of high density lipoprotein cholesterol by the liver. A large body of descriptive information has been published indicating that exercise has a dramatic effect upon hepatic lipid metabolism. The next step in this work is the identification of the molecular mechanisms responsible for these exercise-induced alterations.

Epinephrine-activation of heparin-nonreleasable lipoprotein lipase in 3 skeletal muscle fiber types of the rat.

Epinephrine was used to activate the heparin non-releasable lipoprotein lipase (LPL) in the 3 skeletal muscle fiber types of the perfused rat hindlimb. Following a 9 min washout of the capillary-bound lipoprotein lipase, the hindquarter of the rat was perfused with a buffer containing 10 nM of epinephrine. Activity of the residual LPL in soleus, red vastus lateralis, and white vastus lateralis muscles increased 75%, 96%, and 102% respectively, following epinephrine perfusion. These results suggest that skeletal muscle LPL is under hormonal control possibly through protein phosphorylation by cyclic AMP dependent protein kinase.

Effect of colchicine on alkaline triglyceride lipase activity and triglyceride content in rat skeletal muscle.

One purpose of this study was to determine if colchicine increased intracellular alkaline triglyceride (TG) lipase activity above control levels in rat skeletal muscle. The second aim was to determine the effects of colchicine treatment on the concentration of TG in skeletal muscle. The results show that colchicine was a potent inducer of alkaline TG lipase activity, increasing enzyme activity approximately twofold in slow-twitch red, fast-twitch red, and fast-twitch white muscle types. It was found that in slow-twitch red soleus and fast-twitch red vastus, the two muscle groups with the highest levels of enzyme activity, 76% or more of enzyme activity resides in the intracellular compartment. These results provide evidence that colchicine blocks the export of alkaline TG lipase from skeletal muscle cells similar to that seen in the heart. The finding that TG were reduced at a time when enzyme activity was elevated suggests that intracellular alkaline TG lipase may be playing a role in the hydrolysis of the intramuscular TG droplet.

Role of the alkaline TG lipase in regulating intramuscular TG content.

Three TG lipases have been identified in muscle (i.e., acid, neutral, and alkaline), but as yet we do not know which enzyme is responsible for tissue TG hydrolysis. Over the past 8 yr, work in our laboratory has focused on intracellular lipoprotein lipase (LPL). The results show that this lipase is regulated by the classical cAMP cascade and that the activity of this enzyme is inversely related to endogenous TG concentration. Using these results as a foundation we plan to examine molecular mechanisms involved in the synthesis, compartmentalization, and transport of the alkaline TG lipase. Further, the evidence suggests that this enzyme may be regulated by protein phosphorylation mediated by cyclic AMP-dependent protein kinase. We plan to test this possibility.

Effect of fasting on myocardial substrates in male and female rats.

Adult male and female rats were fasted for 1, 2, or 3 days to determine its effect on circulating and endogenous fuels available to the heart. Liver glycogen was depleted within the first 24 h of food restriction. Plasma glucose decreased approximately 2.5 mM in both sexes during the 3 days. Fasting significantly increased plasma beta-hydroxybutyrate to approximately the same level in female and male rats. Plasma free fatty acid (FFA) increased approximately 0.2 mM in both groups during the first 24 h without food and remained elevated over the next 2 days. FFA concentrations were higher in fed female than in fed male rats and remained significantly higher in female rats throughout the experimental period. Myocardial glycogen increased 64% during the first 2 days of fasting in the male rats and stayed elevated on the third day of fasting. In contrast, heart glycogen of female rats remained unchanged from an initial value of 3.13 mg/g throughout the 3-day fasting period. Endogenous triglyceride (TG) of male rats decreased from 2.14 +/- 0.09 to 1.41 +/- 0.21 mumol/g during the first 24 h without food and remained at that level during the second and third days. Heart TG in female rats fell progressively from 2.36 +/- 0.19 to 1.02 +/- 0.12 mumol/g during the fasting period. Cardiac FFA were higher in female than in male animals throughout the entire experiment. These data indicate that quantitative and qualitative metabolic differences exist between male and female rats stressed by fasting.(ABSTRACT TRUNCATED AT 250 WORDS)

Protein kinase inhibitor blocks the activation of a myocardial triacylglycerol lipase.

Purified inhibitor of the cyclic AMP-dependent protein kinase (PKI) has been used as a probe to determine if hormone and cyclic AMP-induced activation of the cardiac alkaline triacylglycerol (TG) lipase is mediated through the cAMP-dependent protein kinase. Addition of CAM (cyclic AMP, Mg-ATP, and 3-isobutyl, 1-methylxanthine) to any of the four fractions (homogenate, 10,000 g supernatant, 105,000 g supernatant, or heparin-Sepharose eluate) from heparin perfused heart activated the TG lipase 60% to 110%. Preincubation of these fractions with 33 ng of PKI had no effect on control enzyme activity. Addition of PKI (33 ng) to extracts following CAM activation had little effect on homogenate TG lipase activity, but reduced activities in 10,000 g and 105,000 g supernatant fractions to their respective control levels, and inhibited TG hydrolase activity of activated heparin-Sepharose eluate to 50% below the control activity. If extracts were preincubated with PKI prior to CAM addition, TG lipase activity was reduced to approximately 50% below control levels in all fractions. PKI addition (33 ng) to 105,000 g supernatant obtained from hearts stimulated 60% by epinephrine perfusion reduced activity to 50% below the control level. PKI inhibition of TG lipase activity of 105,000 g supernatant could be reversed by adding 0.5 microgram of catalytic subunit of protein kinase (PKC) to the extract. The inhibition below control levels caused by CAM and PKI indicate that the PKI-PKC complex by itself or in combination with other extract molecules, has an inhibitory effect on the TG lipase.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of the triacylglycerol lipase activity in three types of rat skeletal muscle.

The purpose of this study was to characterize the lipolytic activity of the alkaline triglyceride lipase in homogenates of three types of skeletal muscle obtained from heparin-perfused rat hindlimb. Specifically, the red portion of the vastus lateralis, the white portion of the vastus lateralis, and the soleus muscles were examined. To remove capillary-bound lipoprotein lipase from the capillary beds, muscle was perfused with an erythrocyte-free buffer containing 4% albumin, 5 units of heparin/mL, and 7.5 microM adenosine. Adenosine reduced perfusion pressure from 117 +/- 5 to 86 +/- 6 mmHg (1 mmHg = 133.32 Pa), providing evidence for an effective vasodilation. This vasodilation increased the amount of lipoprotein lipase removed from the capillary beds. By the end of the experiment, perfusates were lipoprotein lipase-free. Oxygen supply to the perfused hindlimb appeared adequate as evidenced by similar high energy phosphate values for perfused and contralateral control tissues. For example, in soleus muscle, ATP content was 4.5 +/- 0.6 vs. 4.2 +/- 0.3 mumol/g, ADP concentration was 1.0 +/- 0.2 vs. 1.4 +/- 0.2 mumol/g, and creatine phosphate level was 12.9 +/- 0.7 vs. 11.0 +/- 0.6 mumol/g for perfused and contralateral control soleus, respectively. In addition, K+ output by the hindlimb was negligible, while glycolytic flux of perfused muscle was similar to that measured in control tissue. The findings that triglyceride levels of soleus and red vastus lateralis were decreased suggest that endogenous triglyceride was providing energy for the hindlimb during perfusion. Skeletal muscle triglyceride lipase activity was stimulated by serum and heparin, inhibited by NaCl and protamine, and had a pH optimum of 8.1.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of dietary sugar and of dietary fat on food intake and body fat content in rats.

The long term ingestion of a sugar-rich diet (low fat) caused severe obesity in adult rats. In a separate experiment, the habitual consumption of a fat-rich diet (40% kcal from fat) also caused severe obesity. Severe obesity developed in both groups of animals even though they did not overeat. Voluntary food intake for the sugar-fed rats averaged 28,314 +/- 756 calories/rat per 55 wks which was similar to the value of 28,884 +/- 953 calories/rat per 55 wks for the fat-fed rats. However, both values were lower than that of 32,869 +/- 588 for the control rats eating Purina chow. Despite a lower caloric intake, carcass fat averaged 45 +/- 1% for rats eating the sugar-rich diet and 46 +/- 2% for rats eating the fat-rich diet, but only 33 +/- 2% for rats eating a diet of Purina chow. These results provide evidence that severe obesity can develop in the absence of hyperphagia in animals eating a sugar-rich or fat-rich diet. Finally, a rat model for severe obesity is presented in which carcass fat ranged from 18% (lean) to 61% (severe obesity) using dietary intervention alone at critical stages of the animal's life.

Effect of cholera toxin on triacylglycerol lipase activity and triacylglycerol content of rat heart.

This study was performed to reexamine the effect of cholera toxin on total and intracellular alkaline lipoprotein lipase (LPL) activity in rat heart. In addition, the relationship between intracellular triacylglycerol (TG)lipase activity and TG content of cardiac tissue was determined in cholera toxin treated rats. One intravenous injection of cholera toxin increased total LPL activity significantly above control activity 4 h following treatment. After 16 h, total enzyme activity in hearts of cholera toxin treated rats was 2.4-fold above control levels and remained significantly above the control activity up to the 24-h time point. Intracellular alkaline TG lipase activity was increased 24%, 59%, 2.1-fold, and 2.1-fold above control levels measured 0.5, 8, 16, and 24 h following cholera toxin treatment, respectively. Heart TG content fell significantly following cholera toxin treatment, with a maximal reduction seen 8 h following agent injection. At that time, TG was 0.61 mumol/g, a reduction of 63% below the control concentration of 1.8 mumol/g. A negative relationship between myocardial intracellular TG lipase activity and TG concentration of r = -0.83 was highly significant (P less than 0.001). These findings indicate that cholera toxin injection can increase total cardiac LPL activity and show that 70% of this increased activity is in the intracellular fraction. The highly significant relationship between enzyme activity and TG content support our working hypothesis that the intracellular TG lipase (LPL) is playing a role in regulating cardiac TG content.

Cyclic AMP activation of a triglyceride lipase in broken cell preparations of rat heart.

The effect of CAM [cyclic AMP, Mg-ATP, and 3-isobutyl, 1-methylxanthine (MIX)] on triacylglycerol (TG) lipase activity in extracts from heparin-perfused rat heart was determined. TG lipase activity in homogenate, 10,000g supernatant, 105,000g supernatant, ammonium sulfate supernatant, and the eluate from heparin-Sepharose was increased between 62 and 151% when incubated with a combination of 0.3 mM cyclic AMP, 5 mM MgCl2, and 2 mM ATP. The addition of Mg-ATP + cyclic AMP caused a greater activation of TG lipase in the various fractions than did Mg-ATP + MIX or cyclic AMP + MIX. These results suggest that activation may be mediated by the classical cyclic AMP-protein kinase cascade. Control and CAM-stimulated activities were increased by heparin and inhibited by NaCl and protamine sulfate. In the absence of serum in the assay, the CAM system caused a relatively greater stimulation of lipolytic activity in each fraction compared to when serum was present in the assay. However, the absolute values were 6.1 to 16.3-fold greater with serum in the assay than without serum. In a similar manner, TG lipase activity was stimulated by CAM between 1.75 and 4.26-fold at pH 7.4, and only between 1.62 and 2.51-fold at pH 8.1. However, the absolute values at pH 8.1 were 6.77 to 31.83-fold greater than those seen at pH 7.4. These data demonstrate, for the first time, the cyclic AMP activation of a TG lipase above basal levels in cell-free fractions of rat heart. It is intriguing to speculate that the intracellular fraction of lipoprotein lipase may play a role in the hormonal regulation of cardiac TG lipolysis.

Protein kinase activation of heparin-releasable lipoprotein lipase in rat heart.

An attempt was made to activate the capillary-bound fraction of lipoprotein lipase (LPL) with cAMP-dependent protein kinase catalytic subunit (PKC). Following a 30s washout period, hearts were perfused for 1 min with buffer containing heparin. Medium was collected during the second 30s of heparin perfusion. Addition of PKC+Mg-ATP to this capillary bed perfusate increased LPL activity from 6.84 +/- 0.72 nmol/ml/min to 13.76 +/- 1.12 nmol/ml/min (P less than 0.001). A similar 2-fold increase in activity was observed when results were expressed on a mg protein basis. Removal of serum from, or addition of 1.0M NaCl to, the assay system inhibited PKC-stimulated LPL activity approximately 85%. These results indicate that capillary alkaline LPL can be activated by PKC assayed under experimental conditions free of other TG lipases. Moreover, these findings suggest that the intracellular fraction of LPL can be activated by cAMP and that this activation is mediated through protein phosphorylation by cAMP-dependent protein kinase.

Dietary-induced severe obesity: exercise implications.

A rat model for severe obesity has been developed by feeding the animals a fat-rich, sugar-rich diet. The concentration of fat in the diet was similar to what most Americans consume (about 40% kcal from fat). Calories from sugar was calculated to be 40.6%. As adults, body fat content in these animals averaged 61 or 51%, depending on whether the fat-rich, sugar-rich diet caused hyperphagia. The rate of body fat accretion in these severely obese rats raised in litters of four was estimated to be 1.78 +/- 0.12 (SE) g X d-1 (61% body fat) or 0.9 +/- 0.03 g X d-1 (51% body fat). In contrast, lean rats eating a diet of Purina chow deposited fat at a rate of 0.20 +/- 0.02 g X d-1, resulting in a carcass fat content of 18%. Preliminary evidence based on adult body weights of sugar-fed rats suggests that sucrose alone can cause severe obesity similar to that seen with dietary fat alone. Currently, an attempt is being made to determine how dietary fat and/or dietary sugar work to produce severe obesity. One possibility is that dietary fat in the form of a chylomicron and dietary sugar in the form of a very low density lipoprotein may modify adipose tissue lipoprotein lipase activity. This enzyme acts as a gatekeeper for circulating triglycerides entering the adipocyte. It is our belief that the results obtained will help to lay the groundwork for determination of the role of exercise in weight control.