Cholesterol metabolism in dystrophic mice. II. Altered enzyme activities

322

BBA Report

BBALIP 50305

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Cholesterol metabolism in dystrophic mice. II. Altered enzyme activities Donald

E. Kuhn * and David M. Logan

Depariment

of Biology, York Uniuemity, North York (Canada)

(Received 12 March 1990)

Key words: Enzyme activity; Cholesterol metabolism; Murine dystrophy In a previous study we found that free cholesterol (FC) and cholesterol ester (CE) concen~ations in fit-giycoiytic (FG) muscle tissue from dystrophic mice are significantly higher than normal. This increase is not due to an increased capacity for de novo cholesterol biosynthesis. HMG-CoA reductase (HMGR) (the enzyme which catalyzes the rate limiting step) activity is significantly decreased in dystrophic muscle compared to normal. This decrease is paralleled by an increased capacity for both CE production and hydrolysis, i.e., both Acyi-CoA:choiesteroi acyitransferase (ACAT) activity and the activities of both iysosomai and sarcopiasmic cholesterol ester hydroiases (CEH) are greatly increased. These enzyme changes in dystrophic FG muscle are similar those observed in normal tissues with elevated levels of choiesteroi, which suggests that such changes are not the cause of the altered cholesterol concentrations but are rather the response of the tissue to elevated levels of cholesterol.

Mice with muscular dystrophy (genotype &/dy) exhibit a wide range of tissue alterations and changes in the concentratons of various constituent biochemicals. Lipid changes are particularly extensive with changes in fatty acids, phospholipids, sphingolipids and cholesterol [l-3]. We have previously reported that cholesterol levels in dystrophic FG muscle are significantly higher than normal [4]. In this study we have measured the activity of critical enzymes of cholesterol metabolism to see if the concentration changes can be ascribed to particular enzymatic alterations. We have found that the capacity for de novo cholesterol biosynthesis is significantiy decreased compared to normal, while the capacities for CE production and breakdown are significantly increased. These changes in dystrophic FG muscle most likely derive from, rather than produce the increased cholesterol levels. It now appears more likely that the increased cholesterol concentration is due to an increased uptake or retention of cholesterol. Male 129 B6Fl/Rej dy/dy mice and control littermates were purchased from the Jackson Laboratories (Bar Harbor, ME) and tested at 4-6 weeks of age. All

* Present address: Department of Medicine, University of Colorado Health Sciences Center, Denver, CO, U.S.A. Correspondence: D.M. Logan, Department of Biology, York University, 4700 Keeie St., North York, Ontario, Canada M3J 1P3. 0~5-27~/90/$03.50

mice were kept at room temperature on a 12 h light/dark cycle with a continuous water and food supply. DTT, HMG-CoA, oleoyl-CoA, mevalonolactone, NADPH, DOPC, egg yolk lecithin, HSA (Fatty acid free), BSA, leupeptin, cholesteryl oleate and oleic acid were purchesed from Sigma (St. Louis, MO). ATP, ADP and Tris were obtained from Boeh~nger-Mannheim (Montreal, Quebec). DL-3-Hydroxy-3-methyl[3-‘4C]glutaryl CoA (56.0 mCi/mmol), [l-‘4C]oleoyl coenzyme A (55.0 mCi/mmol), cholesteryl [I-14C]oleate (51.9 mCi/mmol) and [l-‘4C]oleic acid (58.9 mCi/mmol) were from New England Nuclear (Boston, MA) Eastman Kodak silica-G chromatograms were from Fisher Scientific {Toronto, Ontario). All other chemicals were reagent grade and were obtained from standard suppliers. Mice were killed at the mid-point of the light cycle. Fast glycolytic muscle was isolated from superficial sections of normal and dystrophic gastrocnemius muscles as previously described [4]. Muscle tissues were homogenized (7 : 1, buffer: tissue) in buffer containing 50 mM Tris, 100 mM sucrose, 5 mM DTT, 30 mM EDTA, 100 FM leupeptin and 30 mM of either NaF or NaCl (pH 7.2). The homogenate was centrifuged twice (12OOOxg for 10 min and 100000 xl: for 90 min 4” C). The supernatant was removed and the pellet was resuspended in an equal volume of homogenization buffer and centrifuged as above. The supernatants were combined to give the ‘sarcoplasmic’ fraction and the pellets combined to give the ‘membranous’ fraction.

0 1990 Eisevier Science Publishers B.V. (Biomedical Division)

323 Fractions not used immediately were stored at - 70 o C. Greater than 95% of the HMG-CoA reductase and ACAT activities were always found idn the membranous fraction and greater than 95% of the neutral CEH activity (reportedly a sacroplasmic enzyme) was always found in the sarcoplasmic fraction. Microsomal fractions were obtained from normal and dystrophic muscle by centrifugation of crude homogenates at 12000 x g for 30 min (4” C) and centrifugation of supernatant at 100000 X g for 90 min (4” C). The resulting pellet was re-suspended in an equal volume of fresh homogenization buffer, spun at 100000 X g for 90 min and the pellet retained for experimentation. Cholesterol was extracted as previously described [4] and the concentration determined by the method of Rude1 and Morris [5]. HMGR activity was assayed in volumes of 150 ~1 containing 75 ~1 enzyme solution (membranous fraction in 50 mM Tris, 100 PM leupeptin and 5 mM DTT (pH 7.2)) 50 mM Tris, 5 mM DTT, 2 mM NADPH, 10 mM EDTA, 30 mM NaF and 20 PM [gluturyl-3-‘4C]HMGCoA (pH 7.4). Samples were incubated at 37’ C for 60 min and the reaction terminated by the addition of 25 ~1 of 2.5 M HCl and 60 mM mevalonolactone. The resulting mixture was left at room temperature for a minimum of 2 h to ensure lactonization of mevalonic acid. Precipitated protein was pelleted by centrifugation and aliquots of the supematant were streaked onto silica-G chromatograms (9 x 3 cm). The chromatograms were developed in acetone/benzene (1: 1, v/v) and areas of the sheet containing mevalonolactone (R, 0.50.7) and HMG-CoA (RF 0.1-0.3) were cut out and counted. ACAT activity was measured in an assay essentially as described by Mitropoulos et al. [6]. Membranous CEH activity was determined essentially as described by Brecher et al. [7]. Separation of the reaction product from its substrate was based on the procedure of Pittman et al. [B] and product concentration was determined by liquid scintillation. Sarcoplasmic CEH activity was determined as described above for membranous CEH, except 75 ~1 of the sarcoplasmic fraction was used directly in each assay and 0.01 M Tris (pH 7.4) replaced acetate as buffer. Protein concentrations were determined using an SDS adaption [9] of the method of Lowry et al. The activities of crucial cholesterol metabolizing enzymes are shown in Table I. HMGR is the critical regulatory enzyme in cholesterol synthesis. HMGR activity in dystrophic muscle is decreased to 60% of the normal value and this effect is not due simply to decreased substrate affinity (K, = 2.9 I_LMfor HMGR from both normal and dystrophic muscle). The synthesis of cholesterol esters in these tissues is catalysed by ACAT. ACAT activity in dystrophic FG

TABLE I Cholesterol Metabolising Enzymes in Normal and Dystrophic Fast Glycolytic Muscle

Activities were determined as outlined in the text. The values given are the mean + S.E. for eight determinations of each muscle type. The dystrophic values are all statistically different from normal at P < 0.001, except for HMGR where P < 0.01. Enzyme

Normal

Dystrophic N/D

Specific activity (pmol/h per mg protein) HMGR

ACA

membranous CEH

sarcoplasmic CE

25.2k2.0 15.1k1.3 1.7

21.3kl.7 49.9k4.0 0.43

22.3 f 2.0 151.8k14.2 0.15

16.5+ 1.5 142.0+ 15.1 0.12

muscle tissue is increased by 130% compared to normal. Again the K, is unchanged (30 PM) so the altered activity is not due to an altered substrate affinity. Similar results were obtained with membrane fractions enriched 1.5-fold with free cholesterol (i.e., substrate was not limiting). Cholesterol esters are hydrolysed by CEH’s which are found in both membranous and sarcoplasmic fractions. Assay conditions for each differ suggesting they are distinct enzymes. The membranous CEH activity in dystrophic tissue is dramatically increased compared to normal (approx. 7-fold). In this case the use of a vesicular substrate makes K, determination uncertain but adjustment of substrate concentration indicated that substrate was not limiting in either normal or dystrophic assays. The sarcoplasrnic CEH activity in dystrophic FG muscle is also dramatically increased (more than B-fold) compared to normal. The maintenance of appropriate cholesterol levels in various tissues is recognized as an important feature in mammals [lo]. Cholesterol regulation is known to involve various factors including uptake, de novo synthesis and release. Synthesis is primarily regulated by the activity of HMGR, the enzyme catalyzing the rate limiting step in cholesterol biosynthesis. This enzyme, whose activity can be reduced by feedback inhibition, is modulated by a phosphorylation/dephosphorylation mechanism and its activity is usually thought to represent the cells capacity for de novo synthesis. Thus, in normal tissues which contain elevated levels of cholesterol, HMGR is reduced [ll]. In the muscles of dystrophic mice the lipid content is elevated [12] and a significant component of this increase is in the cholesterol fraction [12,13]. Previous studies however, have not identified the cause(s) of this increase. In this study we have looked at key enzymes of cholesterol metabolism in FG tissue, the most affected tissue. Total HMGR is reduced significantly in dystrophic FG tissue. Thus increased de novo synthesis per

324 se seems unlikely to explain the elevated tissue concentration. Rather the decreased enzyme concentration is consistent with a normal reduction in enzyme synthesis and/or an increased rate of HMGR breakdown due to elevated cholesterol. In contrast to HMGR, enzymes involved in cholesterol ester metabolism are increased dramatically in dystrophic muscle. This is probably not surprising since the majority of the increased tissue cholesterol is present as cholesterol esters rather than free cholesterol. By 4 weeks of age, the largest proportional increase in total cholesterol in dystrophic mouse muscle is in the CE fraction, and by 10 weeks the concentration of cholesterol esters exceeds the concentration of free cholesterol whereas they are only a modest fraction in normal muscle. Cholesterol esters are produced in most mammalian cells from FC and fatty acyl-CoA molecules by the action of ACAT. Previous studies have shown that CE production is directly proportional to ACAT activity which is increased in cells which have increased FC levels [14,15]. Our finding that ACAT activity is significantly increased in dystrophic muscle indicates that the capacity for CE production in this tissue is increased compared to normal. It is probable that increased ACAT activity in dystrophic muscle is due to the increased concentration of FC, which stimulates the synthesis of the storage form of cholesterol, i.e., cholesterol esters. Again the enzyme change is consistent with a response to elevated cholesterol rather than its production. Two different enzymes appear to be involved in cholesterol ester hydrolysis. Mammary cells have a cholesterol ester hydrolysing activity associated with lysosomes that expresses maximal activity at acid pHs (pH 4.5-5.0) [7,16]. The membrane CEH activity measured in this study is presumably of lysosomal origin since activity was maximal at pH 4.5 (activity at pH 7.4 is < 5% of that at 4.5), more than 95% of the total activity is present in the membrane fraction, and the apparent K, determined is close to that previously reported for lysosomal CEH. The primary function of lysosomal CEH appears to be the hydrolysis of exogenous CE taken up by the cell as components of lipoproteins [17,18]. Furthermore, lysosomal CEH activity is known to increase when the uptake of such CE is increased [19]. This suggests that the increased lysosomai CEH activity in dystrophic muscle may be due to an increase in CE uptake from the serum in lipoproteins. The increase in free cholesterol in the same tissue might then be due to hydrolysis of this enhanced ester concentration at a rate greater than ACAT can catalyze the reverse reaction. The presence of an enzyme activity (S-CEH) in the sarcoplasm of normal and dystrophic FG muscle which hydrolyzes CE at neutral pH is consistent with previous reports [8,20]. (The activity measured at acidic pHs is

less than 3% of the activity measured at pH 7.4, suggesting that S-CEH is distinct from the membranous CEH). Very little is known about S-CEH regulation in nonsteroidogenic tissues, or of the effects of altered CE levels on expressed activity. However, our results showing an increased activity level in cells containing an increased cholesterol ester concentration and those of Brecher and Chobanian [Zl] are consistent with a positive feedback control mechanism involving CE. The increase in measurable S-CEH, like M-CEH, may contribute to the increase in FC in dystrophic muscle. The dramatic increases in both hydrolases minimize the possibility that the increased concentration of CE is simply due to a decreased capacity for CE hydrolysis. Mammalian cells maintain their cholesterol by de novo synthesis ester hydrolysis and by exchange with the serum [10,22]. Our results suggest that an alteration exists in dystrophic muscle cells in either the uptake or release of cholesterol. An increased net uptake of cholesterol could be due to numerous factors, including an increased number of lipoproteins taken up through receptor-mediated endocytosis, increased non-specific ‘bulk phase’ pinocytosis, increased passive exchange due to an increased plasma to cell chemical potential gradient, deceased membrane integrity, increased amounts of cholesterol in lipoproteins, or decreased clearance from the muscle via HDL and its receptor. The increased proportion of CE to FC in cells containing increased CEH activities suggests the increase in cholesterol may be due to an increased uptake, rather than a decreased clearance from muscle. Furthermore, a significant increase in L-CEH activity in dystrophic muscle cellssuggests an increase in LP-CE uptake via receptor-mediated endocytosis, since it has been reported that L-CEH activity is increased when LP-CE uptake, via the receptor, is increased, but CE taken up by bulk phase or passive exchange mechanisms does not lead to increased L-CEH activity [19]. Our results are, therefore, consistent with the suggestion that increased uptake of LP-CE via receptor-mediated endoytosis is, at least partially, responsible for increased cholesterol levels in dystrophic muscle. However, they do not rule out the possibility that lipoproteins are entering the muscle cells via lesions in the sarcolemma. Our results suggest that the increased cholesterol levels and CE/FC ratio in dystrophic FG muscle are not due to alterations in the cholesterol metabolizing enzymes studies. Rather. the enzyme alterations are most likely the result of tissue response to increased tissue concentrations of cholesterol. Acknowledgement The Natural Canada

research was supported by a grant from the Sciences and Engineering Research Council of to D.M.L.

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