Reduced lipoprotein lipase activity in postural skeletal muscle during aging.

Lipoprotein lipase (LPL) is a key enzyme for fatty acid and lipoprotein metabolism in muscle. However, the effect of aging on LPL regulation in skeletal muscle is unknown. We report the effect of aging on LPL regulation in the soleus (red oxidative postural) muscle and the tibialis anterior (white glycolytic non-weight-bearing) muscle in 4- and 24-mo-old Fischer 344 rats and 18- and 31-mo-old Fischer 344 x Brown-Norway F1 (F-344 x BN F1) rats. Total and heparin-releasable LPL (HR-LPL) activities were decreased 38% (P < 0.01) and 52% (P < 0.05), respectively, in the soleus muscle of the older Fischer 344 rats. There was a 32% reduction (P < 0.05) of total LPL protein mass in the soleus muscle with aging. The results were confirmed in another strain. A decrease of total LPL activity (-50%, P < 0.05) was also found in the soleus muscle between 18- and 31-mo-old F-344 x BN F1 rats. LPL mRNA concentration in the soleus muscle was not different between ages. Total LPL protein mass was reduced by 46% (P < 0.05) in the soleus muscle of the 31-mo-old F-344 x BN F1 rats. In the tibialis anterior muscle, neither LPL activity nor mRNA concentration was affected by age in either strain. In conclusion, LPL regulation in a non-weight-bearing muscle was not affected by aging. However, there was a pronounced reduction in LPL activity and LPL protein mass in postural muscle with aging.     

Short-term incubation of cardiac myocytes with isoprenaline has no effect on heparin-releasable or cellular lipoprotein lipase activity.

Heparin (5 units/ml) produced a rapid (5-10 min) release of lipoprotein lipase (LPL) into the incubation medium of cardiac myocytes. Preincubation of myocytes for 30 min with 0.01-10 microM-isoprenaline, 100 microM-forskolin or 500 microM-8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate did not increase heparin-releasable LPL activity. Incubation with isoprenaline also did not change cellular LPL activity, even though the catecholamine did increase the phosphorylase a activity ratio.     

Interaction of lipoprotein lipase with native and modified heparin-like polysaccharides

1. Lipoprotein lipase (EC 3.1.1.34), which was previously shown to bind to immobilized heparin, was now found to bind also to heparan sulphate and dermatan sulphate and to some extent to chondroitin sulphate. 2. The relative binding affinities were compared by determining (a) the concentration of NaCl required to release the enzyme from polysaccharide-substituted Sepharose; (b) the concentration of free polysaccharides required to displace the enzyme from immobilized polysaccharides; and (c) the total amounts of enzyme bound after saturation of immobilized polysaccharides. By each of these criteria heparin bound the enzyme most efficiently, followed by heparan sulphate and dermatan sulphate, which were more efficient than chondroitin sulphate. 3. Heparin fractions with high and low affinity for antithrombin, respectively, did not differ with regard to affinity for lipoprotein lipase. 4. Partially N-desulphated heparin (40–50% of N-unsubstituted glucosamine residues) was unable to displace lipoprotein lipase from immobilized heparin. This ability was restored by re-N-sulphation or by N-acetylation; the N-acetylated product was essentially devoid of anticoagulant activity. 5. Partial depolymerization of heparin led to a decrease in ability to displace lipoprotein lipase from heparin–Sepharose; however, even fragments of less than decasaccharide size showed definite enzyme-releasing activity. 6. Studies with hepatic lipase (purified from rat post-heparin plasma) gave results similar to those obtained with milk lipoprotein lipase. However, the interaction between the hepatic lipase and the glycosaminoglycans was weaker and was abolished at lower concentrations of NaCl. 7. The ability of the polysaccharides to release lipoprotein lipase to the circulating blood after intravenous injection into rats essentially conformed to their affinity for the enzyme as evaluated by the experiments in vitro.     

Exercise induces lipoprotein lipase and GLUT-4 protein in muscle independent of adrenergic-receptor signaling.

Exercise increases the expression of lipoprotein lipase (LPL) and GLUT-4 in skeletal muscle. Intense exercise increases catecholamines, and catecholamines without exercise can affect the expression of both LPL and GLUT-4. To test the hypothesis that adrenergic-receptor signaling is central to the induction of LPL and GLUT-4 by exercise, six untrained individuals [age 28 +/- 4 (SD) yr, peak oxygen uptake 3.6 +/- 0.3 l/min] performed two exercise bouts within 12 days. Exercise consisted of cycling at approximately 65% peak oxygen uptake for 60 min with (block trial) and without (control trial) adrenergic-receptor blockade. Exercise intensity was the same during the block and control trials. Plasma catecholamine concentrations were significantly higher and heart rates were significantly lower during the block trial compared with the control trial, consistent with known effects of adrenergic-receptor blockade. However, blockade did not prevent the induction of either LPL or GLUT-4 proteins assayed in biopsies of skeletal muscle. LPL was significantly increased by 170-240% and GLUT-4 was significantly increased by 32-51% at 22 h after exercise compared with before exercise during both the control and block trials. These findings provide evidence that exercise increases muscle LPL and GLUT-4 protein content via signals generated by alterations in cellular homeostasis and not by adrenergic-receptor stimulation.     

Increased lipoprotein lipase activity of skeletal muscle in cold-acclimated rats

The activity of lipoprotein lipase (LPL) in the heart, diaphragm, and soleus muscles was markedly increased in cold-acclimated rats and it was even greater in rats treated with oxytetracycline (OTC) while exposed to cold. Other skeletal muscles studied had low and variable activities which were not significantly increased by cold acclimation or by cold plus OTC treatment. It appears therefore that, apart from the heart and the muscles involved in respiratory movements, LPL activity is primarily associated with those muscles which contain a predominance of slow-twitch oxidative fibers, and that the enzyme in muscle, heart, and diaphragm responds to cold acclimation and cold plus OTC treatment in a parallel fashion in these tissues.     

The lipoprotein lipase (clearing-factor lipase) activity of cells isolated from rat cardiac muscle.

The total lipoprotein lipase activity recovered in suspension of cells prepared from adult rat hearts was unaffected by the nutritional state of the animals used. The enzyme activity present in the cell suspensions was almost exclusively associated with the cardiac muscle cells present as the major cell type.     

CL-proteins and the regulation of lipoprotein lipase activity in locust flight muscle

Lipoprotein lipases in the flight muscles of Locusta migratoria show a marked substrate specificity: diacylglycerols associated with the adipokinetic hormone (AKH)-induced lipoprotein, A+, are hydrolysed at 4 to 5 times the rate of those associated with the lipoprotein in resting (non-hormone-stimulated) locusts, Ayellow. To determine the basis for this discrimination, the effect on the activity of flight muscle lipoprotein lipase of CL-proteins, a major constituent of lipoprotein A+, but not of Ayellow, has been investigated; they inhibit the flight muscle enzyme in a competitive manner whether activity is measured with a natural lipoprotein substrate, a lipid emulsion or a water soluble substrate. Experiments in vivo suggest that the flight muscle enzyme is normally inhibited in resting (non-AKH-stimulated) locusts but, interestingly, injection of synthetic AKH-I relieves the inhibition and increases the activity by 30 to 40%. This is not a direct effect of the hormone on the enzyme, but appears to be related to the hormone-induced formation of lipoprotein A+, so that the majority of CL-proteins in the haemolymph become bound to this lipoprotein and the concentration of free CL-proteins is markedly reduced. We suggest that CL-proteins play a major role in the regulation of lipoprotein lipase in locust flight muscle.     

Determination of lipoprotein lipase activity using a novel fluorescent lipase assay

A novel, real-time, homogeneous fluorogenic lipoprotein lipase (LPL) assay was developed using a commercially available substrate, the EnzChek lipase substrate, which is solubilized in Zwittergent. The triglyceride analog substrate does not fluoresce, owing to apposition of fluorescent and fluorescent quenching groups at the sn-1 and sn-2 positions, respectively, fluorescence becoming unquenched upon release of the sn-1 BODIPY FA derivative following hydrolysis. Increase in fluorescence intensity at 37°C was proportional to LPL concentration. The assay was more sensitive than a similar assay using 1,2-O-dilauryl-rac-glycero-3-glutaric acid-(6-methylresorufin ester) and was validated in biological samples, including determination of LPL-specific activity in postheparin mouse plasma. The simplicity and reproducibility of the assay make it ideal for in vitro, high-throughput screening for inhibitors and activators of LPL, thus expediting discovery of drugs of potential clinical value.     

Endurance training has little effect on active muscle free fatty acid, lipoprotein cholesterol, or triglyceride net balances

We evaluated the hypothesis that net leg total FFA, LDL-C, and TG uptake and HDL-C release during moderate-intensity cycling exercise would be increased following endurance training. Eight sedentary men (26 +/- 1 yr, 77.4 +/- 3.7 kg) were studied in the postprandial state during 90 min of rest and 60 min of exercise twice before (45% and 65% V(O2 peak)) and twice after 9 wk of endurance training (55% and 65% posttraining V(O2 peak)). Measurements across an exercising leg were taken to be a surrogate for active skeletal muscle. To determine limb lipid exchange, femoral arterial and venous blood samples drawn simultaneously at rest and during exercise were analyzed for total and individual FFA (e.g., palmitate, oleate), LDL-C, HDL-C, and TG concentrations, and limb blood flow was determined by thermodilution. The transition from rest to exercise resulted in a shift from net leg total FFA release (-44 +/- 16 micromol/min) to uptake (193 +/- 49 micromol/min) that was unaffected by either exercise intensity or endurance training. The relative net leg release and uptake of individual FFA closely resembled their relative abundances in the plasma with approximately 21 and 41% of net leg total FFA uptake during exercise accounted for by palmitate and oleate, respectively. Endurance training resulted in significant changes in arterial concentrations of HDL-C (49 +/- 5 vs. 52 +/- 5 mg/dl, pre vs. post) and LDL-C (82 +/- 9 vs. 76 +/- 9 mg/dl, pre vs. post), but there was no net TG or LDL-C uptake or HDL-C release across the resting or active leg before or after endurance training. In conclusion, endurance training favorably affects blood lipoprotein profiles, even in young, healthy normolipidemic men, but muscle contractions per se have little effect on net leg LDL-C, or TG uptake or HDL-C release during moderate-intensity cycling exercise. Therefore, the favorable effects of physical activity on the lipid profiles of young, healthy normolipidemic men in the postprandial state are not attributable to changes in HDL-C or LDL-C exchange across active skeletal muscle.     

Increasing effect of vanadate on lipoprotein lipase activity in isolated rat fat pads

Vanadate increased lipoprotein lipase (LPL) activity in the isolated fat pads in a time- and dose-dependent manner. The increasing effect of vanadate was inhibited by amiloride, similar to that of insulin, and it also was not additive to that of insulin. Although the increasing effects of vanadate and insulin were preserved in K(+)-free medium, appreciable decreases in both effects were observed by replacement of Na+ with choline ion or omission of Ca2+ in the medium. Vanadate showed the full effect in the presence of cycloheximide at concentrations that inhibited protein synthesis of the fat pads, suggesting that the action of vanadate is not due to the increase in protein synthesis. Tetrakis (acetoxymethyl) ester of quin 2 at 50 microM concentration never inhibited the action of vanadate though it showed a little inhibition at a concentration of 300 microM. No inhibition of the action of vanadate was observed with ruthenium red. These results suggest that vanadate increases the LPL activity via a process less sensitive to the intracellular Ca2+ concentration. Adrenaline, dibutyryl cyclic AMP, and 3-isobutyl-1-methylxanthine all inhibited the action of vanadate, suggesting that the action is inhibited with increase in the intracellular concentration of cyclic AMP. Monensin and carbonyl cyanide m-chlorophenylhydrazone inhibited the action of vanadate. In contrast, the action of insulin was never inhibited by monensin. Tunicamycin and 2-deoxyglucose, at rather high concentrations, inhibited both actions. These findings suggest that vanadate increases the LPL activity through mechanisms of action involving amiloride- and monensin-sensitive pathways dependent on energy.     

Post-translational regulation of lipoprotein lipase activity in adipose tissue.

Changes in adipose-tissue lipoprotein lipase activity that are independent of protein synthesis were investigated in an incubation system in vitro. Under appropriate conditions at 25 degrees C a progressive increase in the enzyme activity occurs that is energy-dependent. Part of the enzyme is rapidly inactivated when the tissue is incubated with adrenaline or adrenaline plus theophylline. The mechanism of this inactivation appears to be distinct from, and to follow, the activation of the enzyme. A hypothesis is presented to account for the results in terms of an activation of the enzyme during obligatory post-translational processing and a catecholamine-regulated inactivation of the enzyme as an alternative to secretion from the adipocyte.     

Exercise training increases ERK2 activity in skeletal muscle of obese Zucker rats.

Acute exercise and training increase insulin action in skeletal muscle, but the mechanism responsible for this effect is unknown. Activation of the insulin receptor initiates signaling through both the phosphatidylinositol (PI) 3-kinase and the mitogen-activated protein kinase [MAPK, also referred to as extracellular signal-regulated kinases (ERK1/2)] pathways. Acute exercise has no effect on the PI3-kinase pathway signaling elements but does activate the MAPK pathway, which may play a role in the adaptation of muscle to exercise. It is unknown whether training produces a chronic effect on basal activity or insulin response of the MAPK pathway. The present study was undertaken to determine whether exercise training improves the activity of the MAPK pathway or its response to insulin in obese Zucker rats, a well-characterized model of insulin resistance. To accomplish this, obese Zucker rats were studied by using the hindlimb perfusion method with or without 7 wk of treadmill training. Activation of the MAPK pathway was determined in gastrocnemius muscles exposed in situ to insulin. Compared with lean Zucker rats, untrained obese Zucker rats had reduced basal and insulin-stimulated activities of ERK2 and its downstream target p90 ribosomal S6 kinase (RSK2). Seven weeks of training significantly increased basal and insulin-stimulated ERK2 and RSK2 activities, as well as insulin stimulation of MAPK kinase activity. This effect was maintained for at least 96 h in the case of ERK2. The training-induced increase in basal ERK2 activity was correlated with the increase in citrate synthase activity. Therefore, 7 wk of training increases basal and insulin-stimulated ERK2 activity. The increase in basal ERK2 activity may be related to the response of muscle to training.     

The effect in vitro of high-density lipoprotein on hydrolysis of triacylglycerol by lipoprotein lipase.

In an incubation system in vitro with fully activated Intralipid as substrate, rat high-density lipoprotein inhibits the hydrolysis of triacylglycerol by lipoprotein lipase from rat adipose tissue, but does not inhibit hydrolysis by the enzyme from bovine milk. The pattern of inhibition suggests that substrate and high-density lipoprotein may compete for association with rat adipose-tissue lipoprotein lipase.     

Developmental changes in the activity of lipoprotein lipase (clearing-factor lipase) in rat lung, cardiac muscle, skeletal muscle and brown adipose tissue.

The lipoprotein lipase activity of the lung, skeletal muscle, heart muscle and brown adipose tissue of the rat was studied during the period from late foetal to adult life. The enzyme activity in all four tissues emerged substantially during the first 24th after birth. Subsequently, heart and lung enzyme activity remained relatively constant per unit wet weight of tissue. The enzyme activity present in brown adipose tissue and skeletal muscle was elevated per unit weight of tissue during suckling compared with other periods of life. Delivery of near-term foetuses stimulated the emergence of enzyme activity in all four tissues with the same time course as that evoked by normal delivery. The significance of the presence of the enzyme in the tissues and the activity changes which occurred during development are discussed in relation to possible mechanisms of control.     

The effect of short-term starvation on the lipoprotein lipase activity of adipose tissue and cardiac muscle during postnatal development of the rat.

When male rats of between 6 and 13 days of age were starved for 6 h the lipoprotein lipase activity of the epididymal and subcutaneous white adipose tissue did not decline as it did in adults and in animals aged 14-30 days. The lipoprotein lipase activity in the hearts of animals from 6 days of age increased in response to starvation as it did in adults. The relationship of these changes to changes in circulating hormone levels during development was considered.