Search

The research report concluded, "It appears that self-reported sleep reported of participants may be associated to the indirect effects of visual and attitudinal cue and concern about property devaluation rather than distance to the nearest WT's or noise as itself." Careful reading of the report shows that the conclusions presented are not supported by the data provided in the report., (Copyright © 2016 Elsevier Inc. All rights reserved.)

HMG-CoA reductase inhibitor drugs or 'statins' have been shown to effectively reduce plasma total cholesterol (CHOL), CHOL associated with low-density-lipoprotein (LDL), and triglycerides (TG). In addition, slight elevations in HDL-CHOL are also typically observed. Poloxamer 407 (P-407), a nonionic surfactant, effectively elevates both plasma CHOL and especially TG in a dose-controlled fashion and results in formation of atherosclerotic lesions in the aortas of C57BL/6 mice without the requirement of dietary cholic acid [1,2]. The purpose of the present study was to assess whether a typical statin, namely atorvastatin (Lipitor(R)) would significantly reduce P-407-induced hypercholesterolemia and hypertriglyceridemia as well as cause regression of atherosclerotic lesions resulting from administration of P-407 to C57BL/6 mice. C57BL/6 mice in the present study were treated with either normal saline (C, controls), 0.5 g/kg of P-407 (P), or a high-fat, high-cholesterol, cholate-containing diet (HF) for 120 days. Mice in all groups were then equally and randomly divided and treated with either atorvastatin or saline for an additional 120 days. Beginning at Day 121 and using mice in groups P and HF as an example, one-fourth of the mice in each group received 20 mg/kg per day of atorvastatin with either concomitant HF feeding or P-407 administration ('progression' treatment groups), one-fourth received 20 mg/kg per day of atorvastatin following cessation of HF feeding or P-407 administration, one-fourth received saline (placebo) with either simultaneous HF feeding or P-407 administration ('progression' placebo groups), and one-fourth received saline (placebo) following cessation of HF feeding or P-407 administration. Total plasma CHOL was significantly (P<0.01) lower for mice in groups P and HF when administered atorvastatin relative to saline, but remained significantly (P<0.05) elevated compared to total plasma CHOL of C mice. With discontinuation of either P-407 administration or HF feeding, total plasma CHOL declined rapidly in both P and HF mice with atorvastatin-treated mice generally demonstrating lower plasma CHOL concentrations relative to saline-treated mice. Total plasma TG was significantly (P<0.01) lower for mice in group P administered atorvastatin relative to saline, but remained significantly (P<0.05) elevated compared to plasma TG of C mice. With discontinuation of P-407 administration, total plasma TG declined rapidly in P mice with atorvastatin-treated mice typically demonstrating lower plasma TG concentrations relative to saline-treated P mice. Aortas of mice treated with 20 mg/kg per day of atorvastatin in both groups P and HF, whether maintained on the HF-diet or treated with P-407 from Day 120 to 240 or whether each treatment was terminated at Day 120, revealed no presence of atherosclerotic lesions relative to saline-treated mice and were indistinguishable from aortas retrieved from C mice. Atorvastatin at a dose of 20 mg/kg per day not only significantly reduced the plasma CHOL and TG concentrations, but also resulted in regression of atherosclerotic lesions induced in C57BL/6 mice by administration of P-407 or ingestion of a HF-diet containing cholic acid.

This study investigated the potential alteration in the amount of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase messenger RNA (mRNA) and lipoprotein lipase (LPL) mRNA in the livers of C57BL/6 mice after long-term (200 days) treatment with the nonionic surfactant called poloxamer 407 (P-407). Previously, P-407 has been used to produce a dose-controlled hyperlipidemic state in C57BL/6 mice with subsequent formation of atherosclerotic lesions. Five groups of mice were studied; controls (C); mice fed a standard chow diet enriched with only cholic acid (CH); mice fed the high-cholesterol, high-fat Paigen diet (HF); mice treated with 0.5 g/kg P-407 every third day (P); and mice administered 0.5 g/kg P-407 every third day while consuming a diet identical to that of mice in group CH (PC). Neither a significant (p < 0.05) weight loss nor alteration in liver enzymes (AST and ALT) were observed for any group throughout the study when compared with the control mice. Total plasma cholesterol (CHOL) was significantly elevated compared with controls for mice in groups HF, P, and PC, whereas total plasma triglycerides (TG) were significantly increased for mice in only groups P and PC. Long-term ingestion of a high-fat diet or a diet enriched in cholic acid resulted in a significant (p < 0.05) reduction in HDL-CHOL when compared with controls. Plasma samples assayed at 200 days for mice in groups HF and P showed a shift in the lipoprotein fraction distribution primarily to VLDL-CHOL as compared with mice in group C in which, as expected, most of the CHOL was contained in the HDL fraction. The biologic activity of HMG-CoA reductase assayed in hepatic microsomal homogenates was significantly reduced for mice in groups CH (p < 0.01), HF (p < 0.01), and PC (p < 0.05), but not for mice in group P, when compared with control. A statistical analysis of the data demonstrated significant (p < 0.05) reductions in the HMG-CoA reductase mRNA levels in hepatic tissue for all treatment groups relative to mRNA levels determined for mice in group C. In contrast, no treatment group demonstrated a significant difference in hepatic LPL mRNA levels when compared with mRNA levels determined for control animals. These data demonstrate that P-407 administration to C57BL/6 mice significantly decreased the amount of HMG-CoA reductase mRNA detected in liver.

Poloxamer 407 (P-407) induces hyperlipidemia in the rat. It was the purpose of this investigation to determine if chronic P-407 administration would produce atherogenic arterial lesions in the C57BL/6 mouse, a strain reported to be susceptible to hyperlipidemia-induced atherosclerotic plaque formation. One injection (i.p.) of P-407 (0.5g/kg) produced hypercholesterolemia in the mouse that peaked at 24 h and returned to control levels by 96 h following treatment. Four groups of mice were maintained: (1) saline injected (C); (2) P-407-injected (0.5g/kg every 3rd day) (P); (3) P-407 injected plus cholic acid in the diet (PC); and (4) mice fed a high cholesterol (CHOL) diet containing cholic acid (HF). Mice from each group were sacrificed following 90, 145, 200, or 300 days of treatment. Plasma lipid concentrations, hepatic CHOL concentrations (145 and 300 day), and aortic atherogenic lesion areas were measured. Plasma CHOL and triglyceride remained at control levels throughout the 300 days in the C group. CHOL of the HF animals plateaued at approximately 225 mg/dl. P-407 produced CHOL concentrations of 600 mg/dl in P mice and 1000-1500 mg/dl in PC animals. There was no lesion formation in C mice. However, by 90 days lesions were present in the three other groups. Size of the lesions progressed through day 300 with the largest lesions (184.33 + 27.99 mu2 x 10(-3)) being present in the PC mice. HF and P animals had lesions of 70.50 + 11.35 and 43.33 + 7.88 mu2 x 10(-3), respectively. This study provides an animal model where atherogenesis has been produced with hyperlipidemia induced using a chemical agent.

A single 300-mg i.p. injection of poloxamer 407 (P-407, also called Pluronic F-127) in rats produces a marked hypercholesterolemia for a minimum of 96 h. The purpose of this investigation was to determine mechanisms by which P-407 causes hypercholesterolemia. The enzyme targeted for investigation is the rate-limiting enzyme in cholesterolgenesis, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase. Injection of P-407 in fasted rats resulted in a significant (p < 0.05) elevation in plasma cholesterol (61.2 +/- 4.2 mg/dl) as soon as 1 h after injection compared with sham-injected controls (50.1 +/- 3.7 mg/dl). Plasma cholesterol (CHO) 24 h after injection of P-407 was 449 +/- 57 mg/dl, with the fastest rate of accumulation occurring from 1 to 12 h (approximately 16.6 mg/dl/h). Over the concentration range of 0-5 mM, P-407 did not appear significantly to affect the activity of HMG-CoA reductase in vitro. However, the enzymatic activity assayed in microsomal fractions isolated from the livers of P-407-injected rats reached a maximum of 262 +/- 42.6 pmol mevalonate/min/mg approximately 15 h after injection, with a subsequent decline to control activity (94.1 +/- 8.7 pmol/min/mg) at approximately 40 h after injection. At 48 h after injection of P-407, the activity of HMG-CoA reductase significantly (p < 0.05) decreased below control values with a mean activity of 9.4 +/- 1.2 pmol/min/mg. The CHO concentrations in hepatic tissue were significantly (p < 0.01) increased at 2 h (3.26 +/- 0.19 mg/g) and 4 h (3.75 +/- 0.38 mg/g) and significantly (p < .01) reduced at 15 h (1.56 +/- 0.19 mg/g) after injection of P-407 compared with tissue CHO concentrations determined in control animals (2.65 +/- 0.18 mg/g). However, the hepatic CHO content appeared to return to control values by 24 h (mean +/- SEM, 2.61 +/- 0.08 mg/g) after injection. These data suggest that the activity of HMG-CoA reductase is regulated by some indirect mechanism(s) after injection of P-407 in rats.

A single 300-mg intraperitoneal injection of poloxamer 407 (P-407) to rats produces a marked hypercholesterolemia for a minimum of 96 hours and increases the activity of hepatic 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase compared with the enzyme activity found in microsomal homogenates of control animals. We attempted to determine whether inhibition of microsomal HMG-CoA reductase by pravastatin sodium would yield similar values for the maximum reaction in velocity (Vmax) and the HMG-CoA reductase-pravastatin dissociation constant (Ki) when the enzyme was in the activated state compared with the control state. Knowledge of the respective values for Vmax and Ki would allow us to determine whether P-407-induced hypercholesterolemia in the rat was refractory to pravastatin treatment. Over a pravastatin concentration range of 0.5-50 nM, enzyme activity in vitro decreased as the drug's concentration increased. A standard Dixon plot of mean values of reciprocal reaction velocity versus pravastatin concentration yielded Ki of 3.7 and 4.1 nM for the control and activated states, respectively. The Vmax for conversion of HMG-CoA to mevalonate in vitro in the presence of pravastatin was 3.5-fold greater when assayed in microsomal homogenates obtained from P-407-injected rats compared with control animals. Dixon plot analysis of the data resulted in Vmax of 58.1 and 202 pmol.min-1.mg-1 for the control and activated states, respectively. These data suggest that whereas the Vmax is affected, injection of P-407 to rats does not alter the binding affinity of pravastatin for receptor(s) contained in HMG-CoA reductase as reflected by similar Ki values. This experimental animal model may be an additional screen with which to rank order the relative potency of HMG-CoA reductase inhibitors by determining the drug's effectiveness when HMG-CoA reductase is in an activated state.

We attempted to determine the mechanism(s) of poloxamer (P)-407-induced hyperlipidemia in rats by administering a lipid-lowering drug with a known mechanism of action. Five weight-matched animals were assigned to each of four treatment groups. Two groups received P-407 300 mg/ml and two received saline 1 ml. One of the P-407 and one of the saline groups were administered nicotinic acid 100 mg/kg by intraperitoneal injection at 6-96 hours after blood sampling. Blood samples were collected at 7 points from time zero to 120 hours and analyzed for triglyceride and cholesterol concentrations. The detergent produces hypertriglyceridemia (HTG) increasing from 53.4 +/- 7.0 mg/dl (time zero) to 4026.9 +/- 42.1 mg/dl by 24 hours. The HTG response was significantly attenuated by nicotinic acid (at t = 24 hrs). This, however, was followed by an average triglyceride concentration increase of 2.8-fold from 72 to 120 hours. The detergent produces a dramatic hypercholesterolemia (HCHO), increasing cholesterol from 47.5 +/- 1.8 mg/dl to 468.5 +/- 27.9 mg/dl by 48 hours. The HCHO was significantly affected by nicotinic acid administration during the accumulation phase. Nicotinic acid reduced cholesterol concentration from 364.4 +/- 16.1 mg/dl to 276.8 +/- 16.4 mg/dl at 24 hours (p < 0.05). It is a potent antilipolytic agent, limiting the free fatty acids available for the synthesis of triglyceride and cholesterol. These data suggest that P-407 may act by stimulating the release of free fatty acids from the adipocyte for at least 24 hours after injection.

A new animal model of hyperlipidemia is being developed using the nonionic surfactant poloxamer 407 (P-407). We investigated the impact of pravastatin on P-407-induced hyperlipidemia. Twenty rats received P-407 300 mg intraperitoneally to induce hyperlipidemia, and 20 control rats received saline injection. Pravastatin was administered orally to an equal number of rats in both groups using three different regimens. A fourth group did not receive pravastatin. At 24 hours after injection, total cholesterol levels in two of the pravastatin groups were 28% and 34% lower than those in animals that did not receive pravastatin (p < or = 0.01). At 48 hours, triglyceride levels were significantly lower in all pravastatin groups (21-44%) versus animals not receiving pravastatin. Pravastatin diminished the effects of P-407 on lipoproteins. This new animal model may be useful in screening for investigational antihyperlipidemic agents.

The presence of lipoprotein lipase (LPL) in L6 muscle cells is equivocal. Analysis of a 21-day time course indicates that these cells express both LPL activity and mRNA. Lipase activity peaked at 4 days after plating and decreased to a nadir at day 21 after plating. Characterization of lipase activity at 4 and 19 days after plating, corresponding to myoblasts and myotubes, respectively, indicated that most of the enzyme activity had the properties of LPL, including an alkaline pH optimum, a serum requirement, and inhibition by NaCl. LPL mRNA expression peaked at 7 days after plating and fell slightly (24%) at day 21. The primary LPL mRNA species in these cells is 3.7 kb in length. Lipase activity and LPL mRNA were highly correlated during the nine course (r = +0.82), suggesting transcriptional regulation of the enzyme. These data clearly demonstrate that L6 cells express LPL during differentiation.

One 300 mg i.p. injection of the nonionic surfactant poloxamer 407 (Pluronic F-127) produces a significant increase above control of both circulating cholesterol and triglyceride (TG) concentrations. The present study was conducted to determine the effect of poloxamer 407 (P-407) on the capacity to hydrolyze circulating TG by lipoprotein lipase (LPL) in an attempt to determine the mechanism of action of P-407. The concentration of TG in the rat following a single 300 mg i.p. injection of P-407 was marked, increasing from 84 +/- 10 to 3175 +/- 322 mg/dL at 24 hr. The maximal rate of TG accumulation (5.74 mg/dL/min) in the plasma of P-407-injected rats occurred between 2 and 4 hr post-injection. In vitro incubation of LPL with P-407 significantly inhibited enzyme activity with an inhibitory concentration at which 50% of the enzymatic activity was lost of approximately 24 microM. Concentrations of P-407 exceeding 350 microM in vitro completely inhibited LPL activity. The effects of P-407 on the enzymatic activity of LPL in post-heparin plasma obtained following a single 300 mg dose of P-407 to rats demonstrated greater than 95% suppression of LPL activity 3 hr post-injection compared with controls. Inhibition of LPL activity was greater than 90% as long as 24 hr following a single i.p. injection of P-407. However, while the heparin-releasable fraction of capillary-bound LPL was inhibited in the plasma, LPL activity significantly increased in cardiac and skeletal muscle in poloxamer-injected animals compared with sham-injected controls. Although there was no significant change in LPL activity in adipose tissue, testes, and lung resulting from P-407 treatment, LPL activity increased by 37% in myocardium, 69% in soleus, and 66% in gastrocnemius muscle in P-407-injected rats when compared with controls. Our studies would suggest that the predominant mechanism by which P-407 induced an increase in circulating TG was by a reduction in the rate at which TG was hydrolyzed due to inhibition of heparin-releasable LPL by the surfactant.

Lipoprotein lipase (LPL) is regulated in a tissue-specific manner; exercise increases LPL activity in muscle at the same time it is reduced in adipose tissue. The purpose of this study was to determine the relationship between LPL activity and LPL mRNA in muscle and adipose tissue in rats exposed to one bout of exercise. Immediately after a 2-h swim, LPL activity [pmol free fatty acids (FFA).min-1.mg tissue-1] in the exercised animals was reduced 43% in adipose tissue (110 +/- 26 to 63 +/- 17) and increased almost twofold in the soleus muscle (203 +/- 26 to 383 +/- 59) compared with sedentary control animals. At the same time, LPL mRNA was reduced 42% in adipose tissue and increased 50 and 100% in the red vastus and white vastus muscles, respectively. Twenty-four hours after the swim, LPL activity had returned to control levels in adipose tissue and the soleus muscle. At hour 24 of recovery, LPL mRNA was still reduced 23% in the adipose tissue of exercised animals but was not significantly different between exercised and control animals in any of the muscle tissues analyzed. Changes in total RNA concentration could not account for the changes in relative LPL mRNA expression. The relationship between LPL enzyme activity and LPL mRNA in muscle and adipose tissue was +0.86 and +0.93 at 0 and 24 h postexercise, respectively. Thus the tissue-specific changes in enzyme activity induced by exercise could be mediated, in part, through pretranslational control.

The purpose of this work was to determine the relationship between lipoprotein lipase (LPL) activity and LPL mRNA in muscle and adipose tissue in fed and fasted rats. In control animals, the correlation between enzyme activity and LPL mRNA for adipose tissue, heart, soleus, fast red vastus lateralis, and fast white vastus lateralis muscle was r = +0.97. Twenty-four hours of fasting increased LPL activity 38% in heart, reduced it 59% in adipose tissue, and had no effect on activity in the three skeletal muscles analyzed. At the same time, relative LPL mRNA concentrations were reduced 25% in adipose tissue and elevated in heart, soleus, red vastus, and white vastus muscles when compared with control concentrations. Prolonging the fast to 6 days was accompanied by a 64% reduction in adipose tissue LPL activity and an increase in the activities of slow-twitch soleus (83%) and fast-twitch red vastus lateralis muscles (193%), with no enzyme activity change in heart or white vastus lateralis muscle compared with values obtained from control fed animals. LPL mRNA concentration was reduced 66% in adipose tissue, increased more than twofold in heart, soleus, and white vastus muscle, and increased threefold in red vastus muscle. Changes in relative LPL mRNA concentration in adipose tissue induced by fasting could, in part, be accounted for by the increases seen in total RNA concentration. The relationships between enzyme activity and LPL mRNA in muscle and adipose tissue were r = 0.97 and 0.77 for 1-day and 6-day fasted animals, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of insulin and glucose on triacylglycerol (TG) metabolism in normal and diabetic isolated skeletal muscle were investigated in this study. Intracellular TG was continuously synthesized and hydrolyzed in both normal and diabetic skeletal muscle. In the absence of insulin and glucose, normal and diabetic skeletal muscle TG content and synthesis were decreased. In contrast, in the presence of insulin and glucose, the normal and diabetic TG contents were unchanged and triacylglycerol synthesis was increased as compared with the respective control values. However, insulin and glucose increased intramuscular TG content to a greater extent than could be accounted for by their stimulation of TG synthesis, indicating that insulin and glucose appear to inhibit TG hydrolysis in diabetic muscle, as well as in normal muscle. In addition, these data suggest that diabetes causes a defect in the ability of insulin and glucose to stimulate TG synthesis, as the increase in diabetic muscle TG synthesis in the presence of insulin and glucose was less than in normal muscle.

1. A triglyceride (TG) lipase is present in whole homogenate and tissue extracts of beef myocardium with characteristics of lipoprotein lipase (LPL); i.e., activity is stimulated by serum, inhibited by NaCl and protamine sulfate, the protein binds to heparin-Sepharose, and the enzyme has an alkaline pH optimum. 2. This TG lipase, eluted from heparin-Sepharose at 0.9-1.0 M NaCl, has an apparent mol. wt of 64 K daltons. Its primary mRNA is 3.7 kb. 3. Expression of LPL mRNA and enzyme activities are in the ratio of approximately 20:8:1 for hearts of mouse, rat and beef, respectively and correlate with r = +0.99.

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.

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.

Little is known about the contribution of plasma free fatty acid (FFA) and intramuscular triacylglycerol (TG) as substrates for energy production during prolonged electrical stimulation of skeletal muscle. The purpose of this study was to investigate the effects of continuous and intermittent electrical stimulation protocols of different intensities on exogenous FFA oxidation, exogenous FFA incorporation into intracellular TG, and intracellular TG content in the isolated in vitro rat flexor digitorum brevis muscle preparation. Muscles were electrically stimulated for 0.5 h continuously at 0.2 Hz or intermittently (30 s on, 60 s off) at 0.2, 0.4, 0.8, and 5.0 Hz while incubated at 37 degrees C in 0.5 mM palmitate-3% bovine serum albumin medium (pH 7.4) in the presence of insulin (100 microU/ml) and glucose (11 mM). Control muscles were frozen immediately after excision or incubated for 0.5 h. At similar frequencies, less exogenous FFA esterification and more exogenous FFA oxidation occurred during continuous than during intermittent stimulation. As the frequency of intermittent stimulation increased, the amount of exogenous FFA esterified decreased and the amount of exogenous FFA oxidized increased. The data also indicate that at least a portion of TG was constantly being hydrolyzed during electrical stimulation. Under stimulation conditions in which exogenous FFA esterification was below the control (resting muscle) level, intramuscular TG content was significantly decreased compared with control TG content values. Thus both plasma FFA and intramuscular TG are substrates for energy production during electrical stimulation. However, the stimulation parameters employed affect the quantities utilized.

The temporal relationships between triacylglycerol (TG) content and TG lipase activity in slow-twitch (STR) and fast-twitch red (FTR) muscles were determined in rats during recovery from a 2-h swim. Immediately after the exercise, plasma free fatty acid (FFA) was elevated and glycogen concentrations were decreased. TG content was decreased 40% in STR muscle and reduced 45% in FTR muscle. The TG concentration of STR muscle increased in a linear fashion throughout recovery so that control levels were reached within the first 24 h after exercise. TG lipase activity of STR muscle was elevated 36% above control immediately after the swim and continued to increase to 84% above control 24 h after the work. In STR muscle there was a net synthesis of TG, while lipase activity was elevated above that measured in muscle of control rats. TG content of FTR muscle remained 45% below control throughout the first 24 h of recovery, and TG lipase activity increased from 26% (P greater than 0.05) greater than control immediately after exercise to threefold above control 24 h after work. All parameters returned to control levels by 48 h of recovery. These data indicated that a net TG synthesis occurs in STR muscle when lipolytic activity is elevated. In FTR muscle, however, a gradual increase in TG lipase activity that occurs during the first 24 h of recovery accompanies a TG concentration well below the control level throughout this same time frame.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

1. The patagialis muscle of three groups of different aged chickens was subjected to passive stretch by placing an inflexible sleeve over the left wing, and the right wing served as the control. 2. Food was removed from the animals 2 days prior to applying passive stretch. After 6 days of passive stretch on young 7-week-old animals, the stretched muscle was 200% larger than the control. 3. During the restriction, control muscle lost 69% of its weight and the stretched muscle maintained its original weight. 4. Changes in muscle DNA and hydroxyproline content were similar to changes in muscle weight but hydroxyproline content lagged changes in muscle weight. 5. Restricted 10-month-old chickens responded to passive stretch with an absolute increase in muscle mass which was accompanied by increases in protein, DNA and hydroxyproline content. 6. Muscle from the 28-month-old chickens did not respond to either 11 days of restriction or passive stretch. 7. The results of the present study indicate that as the chicken grows older, the ability of the muscle to respond to stimuli, stretch and/or starvation, is reduced.

The contribution of intracellular triacylglycerol (TG) as a substrate for skeletal muscle during electrical stimulation is equivocal. Therefore, the purpose of this study was to investigate the effect of electrical stimulation on the TG content in the isolated intact rat flexor digitorum brevis skeletal muscle preparation by use of two different stimulation protocols. Muscles were electrically stimulated for 1 h either continuously at 1 Hz or intermittently (30 s on, 60 s off) at 5 Hz while incubated in 21 degrees C Krebs bicarbonate buffer (pH 7.4) that contained 11 mM glucose. Control muscles were either frozen immediately after excision or incubated for 1 h. TG content was significantly decreased (P less than 0.05) compared with control concentrations in both stimulated muscle groups, with the greatest reduction (60%) occurring after 5-Hz intermittent stimulation. These data indicate that intramuscular TG is hydrolyzed in response to electrical stimulation in the isolated flexor digitorum brevis muscle preparation. In addition, the type of stimulation (higher frequency intermittent vs. lower frequency continuous) employed influences the amount of intracellular TG hydrolyzed.

The present studies examined adenosine and guanosine 3',5'-cyclic monophosphate (cAMP and cGMP) levels in left ventricular tissue of neonatal and adult rats subjected to 3-10 days of abdominal aortic constriction. Left ventricular cAMP levels were elevated after 3 days of pressure overloading in neonatal rats (2,274 +/- 430 pmol/g; mean +/- SE) compared with composite control values (1,280 +/- 124) obtained from sham-operated neonates, sham-operated adults, and aortic-constricted adult groups. cAMP levels declined progressively until, at 10 days after aortic constriction, values were lower (681 +/- 25 pmol/g) than control (1,621 +/- 107). Left ventricular cGMP level was higher in sham-operated neonatal (38 +/- 3 pmol/g) than in sham-operated adult rats (17 +/- 1) at 3 and 10 days postsurgery, but pressure overloading exerted no effect on cGMP measurements. Adenylate cyclase activity in left ventricular tissue homogenate was higher in 3-day sham-operated neonatal (58 +/- 3 pmol X mg protein-1 X min-1) compared with sham-operated adult (10 +/- 1) rats as the result of augmented nonmuscle cell activity. Elevated cAMP values in 3-day, pressure-overloaded neonates occurred despite lower adenylate cyclase activity (44 +/- 2), via degradative modulation (cAMP phosphodiesterase). Guanylate cyclase activity in left ventricular tissue was consistent with prevailing cGMP levels and was not influenced by aortic constriction. The present experiments show that neonatal cardiac enlargement is associated with biphasic alterations in cAMP level which are modulated, at least in part, via degradative reactions.

Measurement of the cAMP-dependent protein kinase "activity ratio" was introduced by Corbin et al. (Corbin, J.D., Soderling, T.R., and Park, C.R. (1973) J. Biol. Chem. 248, 1813-1821) and has been used by a large number of investigators as an index of the activation of this enzyme in the intact cell. This communication reports that with the typical conditions used the dissociation of the protein kinase is not blocked throughout the extraction. This is demonstrated, with glucagon-stimulated rat liver as the example tissue, by the addition of exogenous protein kinase. These data call into question the meaning of results reported using this experimental approach.

Numerous cellular biochemical events caused by hormones are mediated through cyclic AMP. Although many changes occur in the cell during exercise that could be attributed to this nucleotide, little evidence is available implicating it as an important regulator of exercise metabolism. In this investigation it was found that a 60 min bout of treadmill exercise caused a 2.4-fold increase in myocardial cyclic AMP immediately following the work. Rather than the immediate nucleotide hydrolysis that was expected, it was found that the elevated cyclic AMP level remained for approx. 24 h before returning to control levels. Cardiac glycogen fell to 30% of control after work but supercompensated 60% above control within 1 h following exercise. Therefore, cardiac cyclic AMP was elevated at a time when glycogen was being synthesized. Study of the temporal relationship between the exercise-induced increase in cyclic AMP and cyclic nucleotide phosphodiesterase indicated that the work caused an increase in the hearts' capacity to hydrolyze cyclic AMP. Measurement of heart phosphodiesterase at substrate concentrations of 1.0 and 100 microM produced significant increases in enzyme activity immediately following exercise which remained elevated for 48 h and was back to control activity 96 h following work. These data present a potentially fascinating model for the study of the dissociation between cyclic AMP, glycogenesis and elevations in phosphodiesterase activity in the heart.

Prepubertal, pubertal, and postpubertal (28, 45, and 60 days old, respectively) rats of both sexes were run to exhaustion on a motor-driven treadmill to determine whether sexual maturation was involved in the glycogen sparing phenomenon previously reported in the myocardium of female rats receiving high doses of estrogen. Positive work was calculated and was not different in the male and female age-paired rats. Liver glycogen was significantly depleted by 92-98% in male and 96-97% in female exercising rats of all ages. The exercise bout resulted in significant depletion of glycogen in the red and white portions of the vastus lateralis muscle of male and female animals. At 45 and 60 days of age female animals had significantly more glycogen in the red muscle at exhaustion than age-paired males. Myocardial glycogen was significantly depleted by 32, 38, and 41% in prepubertal and pubertal males and prepubertal females, respectively. Pubertal females depleted myocardial glycogen by 19% (P less than 0.05). Postpubertal male animals exhibited a 46% depletion of myocardial glycogen (P less than 0.01), while myocardial glycogen in sexually mature females was not decreased at exhaustion. These data indicate that a myocardial glycogen sparing phenomenon exists in vivo in sexually mature female rats. These data further suggest that the glycogen sparing phenomenon develops in association with sexual maturation.

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)

Running exercise trained rats at either 60 or 76% of their VO2max caused myocardial cyclic AMP phosphodiesterase (PDE) activity to be increased above control levels for at least 24 h following work. Neither training nor the exercise had any effect on the total concentration of calmodulin in heart tissues. The affinity of PDE for cyclic AMP was not changed by the exercise or training. The chelating agent, EGTA, had the same influence on PDE activity regardless of whether it was present in assays of control or exercised heart extract. Km and EGTA results suggest that calcium-bound calmodulin does not account for the higher PDE activity in the hearts of exercised rats. Supernatants from hearts homogenized in the presence of charcoal, to remove nucleotides from the extract, did not eliminate the exercise-associated increase in PDE activity. These results suggest that the elevated activity was not caused by an in vitro nucleotide activation. Preincubation of the enzyme from exercised and control rat hearts with snake venom activated PDE when assays were performed with the low concentration of cyclic AMP (1 microM). Moreover, the activity reached in the extract of exercisers (23.3 pmol X 100 microL-1 X min-1) was significantly greater than the activity found in control hearts (17.59 pmol X 100 microL-1 X min-1). Exercise increases PDE activity in the myocardium of trained rats.(ABSTRACT TRUNCATED AT 250 WORDS)

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)

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.

In this study the effect of exercise training on the adenosine 3',5'-cyclic monophosphate (cAMP) system of rat adipose tissue has been investigated. The basal amount of cAMP for the exercising rats was 0.179 +/- 0.021 nmol/10(6) cells, the same value as for the controls. Phosphodiesterase activities (low and high Km) remained unaffected as a result of the program of treadmill running. Kinetic constants for the low- and high-Km phosphodiesterases revealed that the affinity of the enzymes for substrate (cAMP) was unaltered by physical training. Finally, ethyleneglycol-bis(beta-aminoethylether)-N,N'-tetraacetic acid, possibly through its effect on calmodulin, stimulated or inhibited (depending on concentration) phosphodiesterase activity in the same direction and to a similar extent in extracts of adipose tissue from runners and controls. Taken together, these data clearly demonstrate the exercise training has no effect on the cAMP system of adipose tissue in male rats.

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.

Type L hormone-sensitive lipase (HSL) activity was increased approx. 35% above control in cardiac myocytes incubated for 15 min with 5 nM-adrenaline. Concomitantly. adrenaline-stimulated myocytes had a lower triacylglycerol content, released more non-esterified fatty acid and had a higher intracellular concentration of cyclic AMP than did myocytes incubated without hormone. The lipase activity measured in adrenaline-stimulated and non-stimulated myocytes was stable in acetone/diethyl ether, stimulated by serum and inhibited by NaCl. These properties are consistent with the type L designation of this HSL. The finding that type L HSL is stimulated by adrenaline indicates that the enzyme that is being activated is found in the cell and not associated with an extracellular compartment of the myocardium.

Adenosine 3',5'-cyclic monophosphate (cAMP) concentration and 3',5'-cyclic-nucleotide phosphodiesterase (PDE) activity were measured in skeletal muscle, heart, and liver of rats exposed to 1, 3, 5, and 7 days of cold. Cyclic nucleotide concentration increased in fast-twitch red muscle at the same time that PDE activity was decreasing. Nucleotide concentration and enzyme activity of slow-twitch red muscle were not altered by the cold exposure. The PDE activity of fast-twitch white muscle was elevated approximately 50% above control after 1 and 3 days of cold exposure. By the 5th day in the cold, white muscle PDE activity had returned to control levels and remained there through the 7th day of experimentation. cAMP concentration in hearts of cold-exposed rats was significantly (P less than 0.01) elevated above control at all time points measured. Myocardial PDE activity was elevated above control (P less than 0.05) at 1 and 3 days of cold exposure but returned to control levels by the 5th day in the cold. Hepatic cAMP and PDE activity were elevated above control at all time points analyzed. These data suggest that changes in cyclic nucleotide metabolism play a role in attaining homeostasis during acute cold exposure.

The influence of increasing the in vivo concentration of cyclic AMP on the activity of cyclic nucleotide phosphodiesterase (PDE) in rat heart was investigated. One, three, and five hourly injections of 5.0 mg dibutyryl (Bt2) cyclic AMP significantly increased the activity of PDE in the supernatant fraction of rat heart using 1.0 microM cyclic AMP as the assay substrate concentration. When 100 microM cyclic AMP was used in the assay reaction, increases in enzymes activity were seen following five and eight nucleotide injections. The nucleotide-induced increase in PDE activity was dose dependent. When the five-injection protocol was used, PDE activity remained elevated for at least 4 h, while activity had returned to control levels within this time when two hourly injections were used. The nucleotide stimulation of PDE activity was blocked by cycloheximide. Five hourly infections of Bt2 cyclic AMP increased PDE activity in the liver and fast-twitch red muscle. A reduction in PDE activity in fast-twitch white muscle was seen following nucleotide injections. These findings are consistent with the hypothesis that prolonged elevations in the intracellular concentration of cyclic AMP cause an elevation in myocardial PDE activity. The increased activity seems to be the result of protein synthesis. These data suggest that cyclic AMP contributes significantly in regulating its own metabolism in the 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.

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 (PK) and hormone-sensitive lipase (HSL) were measured at rest in adipose tissue of untrained male rats and in that of animals subjected to a strenuous program of treadmill running. Total amounts of PK activity (decreased from 860 +/- 104 to 474 +/- 53 pmol.min-1. (10(6) cells)-1 (P less than 0.01) as a result of exercise training. At the same time, binding capacity for adenosine 3',5'-cyclic monophosphate (cAMP) was elevated in the runners. These data suggest a functional loss in catalytic activity without a loss in binding capacity. In addition, the results provide evidence that the capacity of PK to activate HSL is reduced in adipocytes of physically trained rats. HSL activity was measured in both adipose tissue slices and isolated adipocytes. The results show that the levels of activity of HSL did not increase as a result of the running program. These results provide evidence that the lipolytic capacity of adipocytes of normal untrained male 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 HSL activity.

1. Adrenaline has a biphasic effect on intracellular lipoprotein lipase activity and on endogenous triacylglycerol content in heparin-perfused heart. 2. A high concentration of adrenaline (1 microM in the perfusion buffer) activated endogenous lipoprotein lipase activity and, at the same time, decreased intracellular triacylglycerol stores. 3. In contrast, a low concentration (0.005 microM-adrenaline) inhibited intracellular lipoprotein lipase activity. Under these conditions, cardiac triacylglycerol content was elevated above control values. 4. Perfusing the heart with high and low concentrations of 3-isobutyl-1-methylxanthine elicited a biphasic effect on endogenous lipoprotein lipase activity and triacylglycerol content similar to that seen with adrenaline treatment. 5. The effect of adrenaline on intracellular lipoprotein lipase activity appears to be mediated by cyclic AMP through protein kinase. 6. A possible role for intracellular lipoprotein lipase in the regulation of endogenous triacylglycerol in rat heart is proposed.

Nuclei isolated from glucagon-stimulated perfused rat livers contained 2-3 times as much protein kinase activity as did nuclei from control animals. In the presence of either the heat-stable inhibitor or the protein kinase regulatory subunit the elevated cyclic AMP-independent enzyme activity from stimulated nuclei was inhibited to an activity equivalent to that found in controls.

Adrenaline, 3-isobutyl-1-methylxanthine (MIX) and dibutyryl cyclic AMP (Bt2 cyclic AMP) stimulated type-L hormone-sensitive lipase (HSL) activity when measurements were made on defatted rat heart powders. These lipolytic agents stimulated the activity of this enzyme in a time- and dose-dependent manner. This activation was reversible, because removal of adrenaline from the perfusate was accompanied by the return of type-L HSL activity to control levels. We have reported [Palmer, Caruso & Oscai (1981) Biochem. J. 198, 159-166] that perfusion with low levels of adrenaline, MIX or Bt2 cyclic AMP reduced type-L HSL activity below control levels when measurements were made in aqueous homogenates. However, in the present study, when activities were measured in acetone/diethyl ether heart powders, all concentrations of these agents studied stimulated enzyme activity, and at no concentration was there enzyme inhibition. These data suggest that acetone/diethyl ether treatment may remove a factor that plays a role in type-L HSL regulation. Type-L HSL activity measured in acetone/diethyl ether powders of control and stimulated rat heart exhibited properties that include alkaline pH optimum, serum requirement, activation by heparin and inhibition by high salt and protamine sulphate. These characteristics, in addition to the stability of the enzyme to treatment with organic solvents, fulfil the requirements for the type-L HSL classification.