Search

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.

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.

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.

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)

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.

In response to a program of daily swimming for 16 weeks, the activities of pyruvate kinase and lactate dehydrogenase increased significantly in the hearts of young male rats. The isozyme composition in M of cardial lactate dehydrogenase increased from 28.5 to 32.7% in the trained animals. Phosphofructokinase activity and glycogen content were unchanged. The hearts of the exercising animals were 28% heavier than those of sedentary paired weight controls.

The long-term ingestion of a fat-rich diet caused severe obesity in adult rats. Severe obesity developed in these animals without them having to increase caloric intake. Evidence for this comes from the observation that voluntary intake averaged 36,113 +/- 410 calories/rat per 60 wk for rats eating a high-fat diet (42% of calories from fat) compared to a value of 36,125 +/- 500 calories/rat per 60 wk for those eating a diet of Purina chow. Despite a similar caloric intake, carcass fat averaged 51 +/- 1% for rats eating the fat-rich diet but only 30 +/- 1% body fat for control animals eating the low-fat diet of Purina chow. These results indicate that a fat-rich diet does not always cause rats to overeat. Further, they clearly demonstrate that severe obesity can develop in the absence of hyperphagia provided the animals eat a fat-rich diet. Finally, a correlation coefficient of r = 0.76 between body weight and lean body mass was obtained for the Purina chow-fed rats and that of r = 0.66 was obtained for the fat-fed rats.

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.

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)

Hormone-sensitive lipase activity was measured in adipocytes of rats subjected to a 12-wk program of treadmill running. Enzyme activity in the runners sacrificed immediately after exercise increased 2.5-fold (P less than 0.001) in tissue exposed to epinephrine and threefold (P less than 0.001) in tissue not exposed to epinephrine, when the results were expressed per gram of adipose tissue. Increases of almost the same magnitude were observed in runners sacrificed 24 h after their last bout of work. These significant increases in enzyme activity, however, were the result of a significant reduction in the size of cells in the epididymal fat pads of the exercisers compared with those of the freely eating sedentary animals (68.7 +/- 2.7 mum vs. 82.0 +/- 2.7 mum; P less than 0.01). When the results were expressed on a per-cell basis, therefore, hormone-sensitive lipase activity, assayed in the presence or absence of epinephrine, was unaffected by the exercise program. These results provide evidence that the lipolytic capacity of adipocytes of normal, untrained rats is sufficiently large to meet the increased demand for free fatty acids imposed by the exercise program without the need for an adaptive increase in enzyme activity.

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.

Lipoprotein lipase activity was measured in the three skeletal muscle fiber types of untrained rats and in those of rats subjected to a 12-wk program of treadmill running. Lipoprotein lipase activity in slow-twitch red fibers was approximately 14- to 20-fold higher (P less than 0.001) than that in fast-twitch white and approximately 2-fold higher (P less than 0.001) than that in fast-twitch red fibers in the untrained animals. These results suggest that, in sedentary animals, mainly slow-twitch red and fast-twitch red fibers are capable of taking up plasma triglyceride fatty acids. Regularly performed endurance exercise resulted in significant increase (2- to 4.5-fold) in lipoprotein lipase activity in the three muscle fiber types examined. The increase in lipoprotein lipase activity in response to treadmill running suggests that exercise increases the capacity of these fibers to take up and oxidize plasma triglyceride fatty acids. Cardiac muscle did not undergo an exercise-induced increase in the levels of activity of lipoprotein lipase similar to that seen in skeletal muscle.

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.

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.

Lipoprotein lipase (LPL) exists in two distinct fractions in heart and skeletal muscle: LPL in capillary beds regulates the metabolism of chylomicrons and very low-density lipoproteins on the surface of the endothelial cells; in contrast, the intracellular fraction of LPL regulates endogenous triacylglycerol (TG) stores. The name of the intracellular enzyme has been changed from LPL to type L hormone-sensitive lipase (HSL) because it is responsive to epinephrine and glucagon levels in heart. In this symposium evidence will show that epinephrine also activates type L HSL in skeletal muscle. Further justification for the name change is that plasma lipoproteins do not exist in parenchymal cells of muscle and the intracellular enzyme possesses many of the classical characteristics described for LPL. Exercise activates type L HSL in heart and skeletal muscle with a concomitant decrease in muscle TG stores. These results provide evidence that under a normal physiological condition, such as exercise, type L HSL participates in the regulation of intramuscular TG stores.